An in vivo study of novel bioactive peptides that inhibit the growth of Escherichia coli

Abstract: We have created a system in which synthetically produced novel bioactive peptides can be expressed in vivo in Escherichia coli. Twenty thousand of these peptides were screened and 21 inhibitors were found that could inhibit the growth of E. coli on minimal media. The inhibitors could be placed into one of two groups, 1‐day inhibitors, which were partially inhibitory, and 2‐day inhibitors, which were completely inhibitory. Sequence analysis showed that two of the most potent inhibitors were actually peptide?protein chimeras in which the peptides had become fused to the 63 amino acid Rop protein which was also contained in the expression vector used in this study. Given that Rop is known to form an incredibly stable structure, it could be serving as a stabilizing motif for these peptides. Sequence analysis of the predicted coding regions from the next 10 most inhibitory peptides showed that four of the 10 peptides contained one or more proline residues either at or very near the C‐terminal end of the peptide which could act to prevent degradation by peptidases. Collectively, based on what we observed in our screen of synthetic bioactive peptides that could prevent the growth of E. coli and what has been learned from structural studies of naturally occurring bioactive peptides, the presence of a stabilizing motif seems to be important for small peptides, if they are to be biologically active.     

Addition and omission analogs of the 13-residue antibacterial and hemolytic peptide PKLLKTFLSKWIG: structural preferences,model membrane binding and biological activities

The consequences of selective addition or deletion of polar amino acids in a 13-residue antibacterial peptide PKLLKTFLSKWIG on structure, membrane binding and biological activities have been investigated. The variants generated are (a) S and T residues replaced by K, (b) S and T residues deleted individually and together, (c) introduction of two additional K and (d) deletion of L and L with T. In the aqueous environment all the peptides were unordered. In trifluoroethanol, the spectra of peptides belonging to groups (a-c) suggest distorted helical conformation. Peptides in group (d) appear to adopt β-sheet conformation. The peptides bind to zwitterionic and negatively charged lipid vesicles, although to different extents. With the exception of peptides in group (d), all the other peptides exhibited comparable antibacterial activity against Escherichia coli and Staphylococcus aureus. However, the changes made in the peptides in groups (a-c) resulted in reduction of hemolytic activity compared to the parent peptide. Extent of binding to lipid vesicles composed of phosphatidylcholine and cholesterol appears to correlate with hemolytic activity. It appears that polar and charged residues play a major role in modulating the biological activities of the 13-residue peptide PKLLKTFLSKWIG. The 11-residue peptide-like PKLLKFLKWIG has selective antibacterial activity. Thus, by judicious engineering it should be possible to generate short peptides with selective antibacterial activity.     

Not only the nature of peptide but also the characteristics of cell membrane determine the antimicrobial mechanism of a peptide

Abstract: The mechanisms of antimicrobial actions of magainin 2, buforin II and poly l -lysine against various Escherichia coli strains were studied. Poly l -lysine inhibited BL21, ad 434 and GroE⁺/DnaK⁺ growth without lysing the cell. Magainin 2 had a pore-forming activity on BL 21 and ad 434 membrane but could not inhibit the GroE⁺/DnaK⁺ growth in a nutrient-rich medium. Buforin II, which killed BL21 and ad 434 without cell membrane damage, lysed GroE⁺/DnaK⁺ to death. Once they were introduced into the cell by electroporation, all three peptides were able to inhibit cell growth at concentrations of 10 times lower than their MICs. These results indicate that the nature of the peptide and also the characteristics of the cell membrane determine the antimicrobial actions of a peptide.     

New indolicidin analogues with potent antibacterial activity*

Abstract: Indolicidin is a 13‐residue antimicrobial peptide amide, ILPWKWPWWPWRR‐NH₂, isolated from the cytoplasmic granules of bovine neutrophils. Indolicidin is active against a wide range of microorganisms and has also been shown to be haemolytic and cytotoxic towards erythrocytes and human T lymphocytes. The aim of the present paper is two‐fold. First, we examine the importance of tryptophan in the antibacterial activity of indolicidin. We prepared five peptide analogues with the format ILPXKXPXXPXRR‐NH₂ in which Trp‐residues 4,6,8,9,11 were replaced in all positions with X = a single non‐natural building block; N‐substituted glycine residue or nonproteinogenic amino acid. The analogues were tested for antibacterial activity against both Staphylococcus aureus American type culture collection (ATCC) 25923 and Escherichia coli ATCC 25922. We found that tryptophan is not essential in the antibacterial activity of indolicidin, and even more active analogues were obtained by replacing tryptophan with non‐natural aromatic amino acids. Using this knowledge, we then investigated a new principle for improving the antibacterial activity of small peptides. Our approach involves changing the hydrophobicity of the peptide by modifying the N‐terminus with a hydrophobic non‐natural building block. We prepared 22 analogues of indolicidin and [Phe^4,6,8,9,11] indolicidin, 11 of each, carrying a hydrophobic non‐natural building block attached to the N‐terminus. Several active antibacterial analogues were identified. Finally, the cytotoxicity of the analogues against sheep erythrocytes was assessed in a haemolytic activity assay. The results presented here suggest that modified analogues of antibacterial peptides, containing non‐natural building blocks, are promising lead structures for developing future therapeutics.     

Peptide Transporter Function and Prolidase Activities in Caco-2 Cells: A Lack of Coordinated Expression

Abstract

Peptide transport and prolidase activities were measured to determine whether the expression of these two components of protein nutrition are coordinately regulated; i.e., whether an increase in the peptide transporter function will necessarily lead to a higher prolidase activity, or vice versa. The results indicated that peptide transporter function and prolidase activity respond differently to cell differentiation and feeding schedules. The results also indicated that peptide transport and prolidase activities were different in two Caco-2 cell “clones”, with S-K cells transported peptides at higher rates but had lower total prolidase activities, when compared to ATCC cells. These results suggest that the expression of the peptide transporter function and prolidase activity is not coordinated. In addition, both the transporter and the prolidase activities affected the overall transport of Phe when given as the dipeptide Phe-Pro, supporting the notion that intestinal absorption of peptides is an essential component of amino acid absorption. In conclusion, the evidence suggests that the peptide transporter function and prolidase activity are not coordinately expressed by the human intestinal Caco-2 cells.     

Self-association and solubility of peptides.

Self-associated species of the protected C-terminal tetrapeptide segment of porcine vasoactive intestinal peptide and related short sequences in methylene chloride have been disrupted by adding increasing amounts of dimethylsulphoxide. This structural transition has been monitored following the disappearance of the amide I carbonyl stretching band assigned to strongly intermolecularly hydrogen-bonded molecules (1655–1633 cm^-1) in the infrared absorption spectra. A scale for the propensity of the various peptides to aggregate has been established. Substitution of the asparagine residue by a β-tert.-butylaspartic acid residue in the tripeptide drastically reduces the extent of self-association. The increasing tendency to aggregate shown by these peptides is paralleled by a decrease in their solubility. The impact of these results on the strategy of synthesis of porcine vasoactive intestinal peptide is briefly outlined.     

Antibacterial peptides designed as analogs or hybrids of cecropins and melittin

Eight new analogs of cecropin A, two new analogs of melittin and 30 hybrid peptides containing sequences from cecropins and melittin have been synthesized. The lengths of the peptides have varied from 37 residues (the length of cecropin A) to 18 residues. The peptides have been assayed for lysis of sheep red blood cells and for antibacterial activity against two Gram negative and three Gram positive bacteria. The best analogs of cecropin A maintained the anti-Escherichia coli activity of the parental peptide, and were not lytic for red blood cells. Melittin and its replacement analogs were all lytic for red blood cells, but an analog with transposed segments was not. Several of the hybrid peptides were found to be both non-hemolytic and highly active against all test bacteria. The data were used to define the structural requirements for antibacterial activity.     

Peptides containing 2-aminopimelic acid

Taking advantage of the peptide transport strategy, we have designed and synthesized several new peptides containing 2-aminopimelic acid (Apm), an inhibitor of the diaminopimelate pathway in bacteria: L-LYS-ambo-Apm, ambo-Apm-L-Lys, L-Lys-L-Ala-ambo-Apm, ambo-Apm-L-Ala-L-Lys, L-Ala(CI)-ambo-Apm and ambo-Apm-L-Ala(CI). In the two latter cases, Apm was associated with antibacterial amino acid β-chloro-L-alanine [L-Ala(CI)], an inhibitor of alanine racemase and transaminase B. The peptides displayed weak or no antibacterial activities; nevertheless, those containing L-Ala(CI) had low MIC values in the presence of amino acids restoring protein synthesis. When tested on exponential phase Escherichia coli cells grown in minimal medium, the peptides were without effect or bacteriostatic, but important bacteriolytic effects could be observed, especially for the L-Ala(CI)-containing peptides, when the growth medium was supplemented with specific amino acids. It was demonstrated that the weak or nil effect of the L-lysine-containing peptides was due to a poor uptake.     

The Influence of Alternating Magnetic Field on <Emphasis Type="Italic">Escherichia coli</Emphasis> Bacterial Cells

The effect of a low-frequency alternating magnetic field on the properties of E. coli bacterial cells in an aqueous medium have been studied in vitro. The experiments were performed with a daily grown culture of E. coli HB-101 cells obtained in a liquid LB medium. The samples were prepared by means of turbidity-controlled serial dilutions and then grown on a bactoagar. It is established that E. coli colonies grown upon exposure of a monolayer of cells in the alternating magnetic field had a smaller average diameter than the colonies not treated in the field. The number of cell colonies was increased, which indicated that the treatment of E. coli cells in a low-frequency magnetic field produced stimulation of the cell growth. __________ Translated from Khimiko-Farmatsevticheskii Zhurnal, Vol. 39, No. 8, pp.6 – 8, August, 2005.     

Human Dipeptide Transporter,hPEPTl, Stably Transfected into Chinese Hamster Ovary Cells

Purpose. A cDNA encoding the H⁺-coupled peptide transporter, hPEPTl, has previously been cloned from human ileum (8). The objective of this study was to establish a stably transfected cell line expressing hPEPTl in mammalian cell culture. Methods. The hPEPTl cDNA was subcloned into an expression vector carrying the CMV promoter and a neomycin resistance gene. This vector, pCDNA3-PEPT1, was transiently transfected into several cell lines to identify those capable of expressing PEPT1 transport function. CHO cells were selected and stably transfected with PEPT1 (CHO-PEPT1). Dipeptide transport activity was measured with ³H-Gly-Sar, in the presence and absence of inhibitors. Results. The clonal cell line, CHO-PEPT1, displayed high transport activity. Dipeptide transport was sensitive to pH and specific for dipeptides and other small peptides. Peptidomimetic antibiotics, such as cephalexin, were competitors for peptide transport. Conclusions. The stably transfected cell line, CHO-PEPT1 exhibits enhanced transport over that of cell lines with native expression of PEPT1, and therefore, represents a useful tool for rapid screening of drugs that utilize the peptide transporter in the human intestine for absorption.     

Automated synthesis and use of N-chloroacetyl-modified peptides for the preparation of synthetic peptide polymers and peptide-protein immunogens

A method to incorporate N-chloroacetyl moieties at the amino termini of synthetic peptides using a standard program with an automated peptide synthesizer has been developed. The N-chloroacetyl-modified peptides react well with sulfhydryl containing proteins such as 4-mercaptobutyrimide-modified bovine serum albumin to form stable protein-peptide conjugates. By incorporating cysteine into the synthetic peptide, autopolymerization or cyclization of the synthetic peptide occurs by reaction of the free sulfhydryl with the chloroacetyl group. N-Chloroacetyl-derivatized peptides may be useful as reagents for potential peptide immunogens and vaccines.     

A peptide inhibitor of cholesteryl ester transfer protein identified by screening a bacteriophage display library

We screened a bacteriophage display library of random decapeptides to identify peptide inhibitors of cholesteryl ester transfer protein (CETP). After affinity selection against CETP, bacteriophage-infected Escherichia coli were plated at clonal density and 36 random clones were isolated. Analysis of the relevant portion of the bacteriophage DNA from a group of 12 clones that had a relatively high affinity for CETP revealed that the corresponding amino acid sequences of the displayed peptides exhibited an. Xaa-Arg-Met-Arg-Tyr-Xaa composite motif. Based on those results, decapeptides from this group were synthesized and one of them, DPI (NH₂-VTWRMWYVPA-COOH), inhibited CETP-catalyzed transfer of cholesteryl esters and triglycerides. Amino- and carboxy-terminal truncations of DPI demonstrated that the original decapeptide could be reduced to a pentapeptide without loss of either its ability to bind to CETP or its ability to inhibit CETP-mediated lipid transfer. That pentapeptide, NH₂-WRMWY-COOH (WRMWY, PNU-107368E), binds directly to CETP and its inhibition is consistent with that of a competitive inhibitor of CETP with a K_i of 164 μm . WRMWY or modified versions of this peptide may be useful in studying the interactions between CETP and plasma lipoproteins.     

Synthetic peptide vaccines: palmitoylation of peptide antigens by a thioester bond increases immunogenicity

Synthetic peptides have frequently been used to immunize animals. However, peptides less than about 20 to 30 amino acids long are poor immunogens. In general, to increase its immunogenicity, the presentation of the peptide should be improved, and molecular weight needs to be increased. Many attempts have been made to couple peptide immunogens to different carrier proteins [e.g. keyhole limpet haemocyanin (KLH) or ovalbumin]. This leads to very complex structures, however. We used a controlled conjugation of a peptide to a single long-chain fatty acid like palmitic acid by a thioester or an amide bond. It was found that these S-palmitoylated peptides were much more immunogenic than N-palmitoylated peptides and at least similar to KLH-conjugated peptides with respect to appearance and magnitude of induced antibodies (canine parvovirus) or immunocastration effect (gonadotropin-releasing hormone). For chemical synthesis of thioesters, we established conditions for solution and solid-phase synthesis. In both phases, Cys(SBu^t) could only be deprotected efficiently using phosphines, and S-acylation was accomplished using standard coupling at pH 5. We speculate that, in vivo, the presence of an appropriate fatty acid chain, chemically linked through a labile thioester bond, greatly enhances immunogenicity, because it represents a favourable substrate for cleavage by cellular thioesterases in cells of the immune system.     

Renal Assimilation of Short Chain Peptides: Visualization of Tubular Peptide Uptake

Purpose. Renal assimilation of short chain peptides plays an important role in systemic protein metabolism and amino acid homeostasis. The transepithelial peptide transport across the apical membrane of tubular cells is mediated almost exclusively by pH-dependent H⁺-peptide symport pathways. The current study was designed to identify by visualization functional peptide transport activity along the nephron structures. Methods. Visualization of peptide uptake was achieved by using the fluorescent dipeptide derivative D-Ala-Lys-AMCA and unlabelled cefadroxil and glycylglutamine as transport competitors to demonstrate specificity. To confirm these assays, rat specific cRNA probes were synthesized and non-isotopic high-resolution in-situ-hybridization and northern blot analysis were carried out to demonstrate the expression of the high-affinity peptide transporter PEPT2. Results. The reporter molecule was accumulated by cells of the proximal tubulus but not in glomerular or endothelial cells. Inhibition studies revealed competitive inhibition of D-Ala-Lys-AMCA uptake by the betalactam cefadroxil and the dipeptide glycylglutamine. The control organs intestine and spleen did not show uptake of the systemically administered molecule. Non-isotopic mRNA in-situ-hybridization, using an antisense probe for rat PEPT2 confirmed uptake assays by identifying PEPT2 expression throughout segments of the straight proximal tubule at the inner cortex and outer stripe. Conclusions. We demonstrate for the first time renal in vivo transport activity of a dipeptide that allows cells that participate in peptide reabsorption to be visualized. This functional assay may be used to investigate renal peptide transport mechanisms and test new compounds that are transported via proton-driven peptide transporters.     

Testing nanoeffect onto model bacteria: Impact of speciation and genotypes

The gram-negative bacteria Escherichia coli (E. coli) is a very useful prokaryotic model for testing the toxicity of ZnO nanoparticles (nano-ZnO). This toxicity is often linked to Zn²⁺ released from nanoparticles in the culture medium, and nano-ZnO dissolution in different media is clearly established. Here, two model E. coli strains MG1655 and W3110 both descendant from the original K-12 showing slight differences in their genome were submitted to nano-ZnO or Zn²⁺ in order 1?> to refine the nano-ZnO toxicity mechanisms to E. coli, and 2?> to investigate whether toxicity resulted from a real “nanoparticle” effect or from the release of Zn²⁺ in solution. To do so, both strains were submitted to various concentrations (i.e., 0.1–1?mM) of nano-ZnO or Zn²⁺ in Luria Bertani (LB) medium. These toxicity studies take into account the nano-ZnO solubility in the culture medium by specifically monitoring the Zn²⁺ release in our experimental systems. In our experimental conditions, differences in tolerance to nano-ZnO or Zn²⁺ between both strains were clearly evidenced. W3110 is generally more tolerant to metal than MG1655, the latter showing no real difference in its sensitivity to the two zinc added forms unlike W3110. The differences in behavior between both strains could be attributed to differences in the two genomes as a mutation named “amber” in W3110. Moreover, by using these two closely E. coli strains, a real “nano” effect is here clearly demonstrated providing a model to study the toxicity of ZnO nanoparticles.     

Tertiary peptide bond containing-oligo(Leu)s

In order to elucidate the role of tertiary peptide bonds in the conformational development and solubility improvement of peptides, the conformational properties of oligo(Leu)s with the Pro residue and the Gly-(Dmob)Leu sequence were investigated in solution using i.r. absorption, CD, and molar rotation measurements. The i.r. absorption spectroscopy indicated that the peptides soluble in inert solvents such as CCl₄ and toluene had a predominantly β-sheet structure in these solvents. The conformations of the peptides in CCl₄ and toluene were essentially the same as those in a solid state, whereas in THF and in MeOH, the peptides examined were efficiently subjected to solvation, and a randomly coiled structure was predominant. In order to confirm the randomly coiled structure, measurements have been made of the molar rotations of the peptides in a variety of strong proton acceptor and donor solvents. CD measurements are also carried out in MeOH. Through the investigations, it was shown that the protection of peptide bonds and the insertion of the Pro residue had the same effect on conformational and solubilizing behaviors and induced onset of an unordered structure and easy solvation of the peptides in medium and high polarity-solvents.     

Structural and charge requirements for antimicrobial and hemolytic activity in the peptide PKLLETFLSKWIG,corresponding to the hydrophobic region of the antimicrobial protein bovine seminalplasmin

Several analogs of the 13-residue antimicrobial and hemolytic peptide PKLLETFLSKWIG (SPF), which is the most hydrophobic region of the 47-residue antimicrobial protein seminalplasmin [Sitaram, N. & Nagaraj, R. (1990) J. Biol. Chem. 265, 10438-104423 have been synthesized. The antimicrobial and hemolytic properties of the peptides were investigated with a view to gain an insight into the structural and charge requirements for these activities of SPF. Peptides in which E was replaced by K exhibited considerably improved antimicrobial activity with no concomitant increase in hemolytic activity. A peptide in which the aromatic amino acids were replaced by leucine exhibited antimicrobial activity like those of the peptides which had aromatic amino acids. Interchange in the positions of E and K and total replacement of K by E resulted in complete loss of activity. The peptides having antimicrobial activities showed appreciable helical content in a hydrophobic environment, whereas inactive peptides did not. Thus, by suitable‘engineering’ the biological activity of a short 13-residue peptide can be altered to yield peptides specifically having only antimicrobial activity with increased potency. © Munksgaard 1995.     

Studies on the structure of a novel peptide antibiotic,K-582

A novel peptide antibiotic, K-582, which exhibited significant growth inhibition of Candida, viruses and ascites tumor in mice, was found in the culture medium of a strain of Metarhizium anisopliae by Kondo et al. (J. Antibiotics 33 , 535–542 (1980)]. K-582 consisted of two components, designated K-582 A and K-582 B. Threonine, tyrosine, ornithine, and an unusual amino acid were common in both peptides, but lysine was an extra component of K-582 A. The unusual amino acid was identified to be threo-γ-hydroxy-L-arginine (OHArg) by means of mass, nuclear magnetic resonance and infrared spectrometries of the derivatives and the related compounds. The threonine and the arginine were assigned to be L-configuration, and the ornithine and the tyrosine to be D-configuration in both K-582 A and K-582 B, and the lysine to be L-configuration by comparison of their optical rotatory dispersion spectra with those of standard amino acids. The elucidation of primary structure revealed that they were closely related heptapeptides with the following sequence: K-582 A:H-Arg-OHArg-Orn-Thr-Orn-Lys-Tyr-OH; K-582 B:H-Arg-OHArg-Orn-Thr-Orn-OHArg-Tyr-OH, and had the identical sequence in terms of the configuration of their constituents, namely L-L-D-L-D-L-D.     

Influence of protein transduction domains on intracellular delivery of macromolecules

As the plasma membrane and blood–brain barrier selectively restrict the entry of most compounds into cells to < 500 Da, delivering macromolecules into cells was, until recently, little more than a goal. However, with significant effort to capitalise on therapeutic targets available in the post-genomic era, novel approaches for delivering therapeutic macromolecules are being rapidly developed. The discovery of small cationic peptides, termed peptide/protein transduction domains or cell-penetrating peptides, which cross biological membranes, has emerged as a venerable Trojan horse to transport large, biologically active molecules, such as peptides, proteins and oligonucleotides, into mammalian cells in vitro, as well as in preclinical models and clinical trials in vivo. This review discusses the implications of peptide/protein transduction domain-mediated delivery of macromolecules and their possible uses as important primary drug delivery agents.     

Design of chimeric peptide ligands to galanin receptors and substance P receptors

Several chimeric peptides were synthesized and found to be high-affinity ligands for both galanin and substance P receptors in membranes from the rat hypothalamus. The peptide galantide, composed of the N-terminal part of galanin and C-terminal part of substance P (SP), galanin-(1-12)-Pro-SP-(5-11) amide, which is the first galanin antagonist to be reported, recognizes two classes of galanin binding sites (K_D(1)<0.1 nM and K_D(2)∼ 6 nM) in the rat hypothalamus, while it appears to bind to a single population of SP receptors (K_D∼ 40 nM). The chimeric peptide has higher affinity towards galanin receptors than the endogenous peptide galanin-(1-29) (KD ∼ 1 nm ) or its N-terminal fragment galanin-(1-13) (K_D∼ 1,nm ), which constitutes the A′-terminus of the chimeric peptide. Galantide has also higher affinity for the SP receptors than the C-terminal SP fragment-(4-11) amide (K_D= 0.4μm ), which constitutes its C-terminal portion. Substitution of amino acid residues, which is of importance for recognition of galanin by galanin receptors, such as [Trp²], in the galanin portion of the chimeric peptide or substitution of ([Phe⁷] or [Met¹¹]-amide) in the SP portion of chimeric peptide both cause significant loss in affinity of the analogs of galantide for both the galanin- and the SP-receptors. These results suggest that the high affinity of the chimeric peptide, galantide, may in part be accounted for by simultaneous recognition/binding to both receptors. In line with this suggestion is the finding that the binding of the chimeric ligands to the galanin receptor is strongly influenced by the presence of SP (1 μm ) or spantide (1 μm ). We have performed the synthesis and binding studies with 11 chimeric peptides, all composed of the N-terminal galanin-(1-13) fragment or of its analogs, linked to the C-terminal portion of SP or its peptide antagonist, spantide. Our results, similar to earlier reports on chimeric peptides, suggest that high-affinity ligands to peptide receptors can be produced by linking biologically active N-terminal and C-terminal portions of peptides via linkers, enabling a) independent recognition of the chimeric peptide by the relevant receptors and b) intramolecular interactions between the joined N- and C-terminal peptide fragments. These two phenomena may also explain why some of the chimeric peptides have higher affinity than the endogenous peptide(s) and why galantide, and some of its analogs presented here, behave(s) as a galanin receptor antagonist(s).