Role of Cationic Side Chains in the Antimicrobial Activity of C18G.
ABSTRACT: Antimicrobial peptides (AMPs) have been an area of great interest, due to the high selectivity of these molecules toward bacterial targets over host cells and the limited development of bacterial resistance to these molecules throughout evolution. The peptide C18G has been shown to be a selective, broad spectrum AMP with a net +8 cationic charge from seven lysine residues in the sequence. In this work, the cationic Lys residues were replaced with other natural or non-proteinogenic cationic amino acids: arginine, histidine, ornithine, or diaminopropionic acid. These changes vary in the structure of the amino acid side chain, the identity of the cationic moiety, and the pKa of the cationic group. Using a combination of spectroscopic and microbiological methods, the influence of these cationic groups on membrane binding, secondary structure, and antibacterial activity was investigated. The replacement of Lys with most other cationic residues had, at most, 2-fold effects on minimal inhibitory concentration against a variety of Gram-positive and Gram-negative bacteria. However, the peptide containing His as the cationic group showed dramatically reduced activity. All peptide variants retained the ability to bind lipid vesicles and showed clear preference for binding vesicles that contained anionic lipids. Similarly, all peptides adopted a helical conformation when bound to lipids or membrane mimetics, although the peptide containing diaminopropionic acid exhibited a decreased helicity. The peptides exhibited a wider variety of activity in the permeabilization of bacterial membranes, with peptides containing Lys, Arg, or Orn being the most broadly active. In all, the antibacterial activity of the C18G peptide is generally tolerant to changes in the structure and identity of the cationic amino acids, yielding new possibilities for design and development of AMPs that may be less susceptible to immune and bacterial recognition or in vivo degradation.
Project description:Amphiphilic alpha-helices are common motifs used in numerous biological systems including membrane channels/pores and antimicrobial peptides (AMPs), and binding proteins, and a variety of synthetic biomaterials. Previously, an amphiphilic peptide with lysine-containing motifs was shown to reversibly bind the anionic porphyrin meso-Tetra(4-sulfonatophenyl)porphyrin (TPPS4 2-) and promote the formation of excitonically coupled conductive J-aggregates. The work presented here focuses on the use of this amphiphilic peptide and derivatives as a potential antimicrobial agent. AMPs are naturally occurring components of the innate immune system, which selectively target and kill bacteria. Sequence derivatives were synthesized in which the position of the Trp, used as a fluorescence reporter, was changed. Additional variants were synthesized where the hydrophobic amino acids were replaced with Ala to reduce net hydrophobicity or where the cationic Lys residues were replaced with diaminopropionic acid (Dap). All peptide sequences retained the ability to bind TPPS4 2- and promote the formation of J-aggregates. The peptides all exhibited a preference for binding anionic lipid vesicles compared to zwitterionic bilayers. The Trp position did not impact antimicrobial activity, but the substituted peptides exhibited markedly lower efficacy. The Dap-containing peptide was only active against E. coli and P. aeruginosa, while the Ala-substituted peptide was inactive at the concentrations tested. This trend was also evident in bacterial membrane permeabilization. The results indicate that the amphiphilic porphyrin binding peptides can also be used as antimicrobial peptides. The cationic nature is a driver in binding to lipid bilayers, but the overall hydrophobicity is important for antimicrobial activity and membrane disruption.
Project description:Obtaining an in depth understanding of the arms races between peptides comprising the innate immune response and bacterial pathogens is of fundamental interest and will inform the development of new antibacterial therapeutics. Many cationic antimicrobial peptides (AMPs) share a range of structural and physical features that have been linked to antibacterial activity and yet they vary dramatically in their potency towards the same bacterial target. We hypothesised that a whole organism view of AMP challenge on Escherichia coli could provide a sophisticated, bacterial perspective enabling understanding of how potency is linked to mode of action. We used a 1H NMR metabolomic approach to characterise the effect on E. coli of challenge with four structurally and physically related AMPs: magainin 2, pleurocidin, buforin II and a designed peptide comprising D-amino acids only. Sub-inhibitory conditions, where these peptides nevertheless induced a bacterial response, were identified enabling electron microscopic and transcriptomic analyses. Although some common features of the bacterial response to AMP challenge could be identified, the metabolomes, morphological changes and the vast majority of the changes in gene expression were specific to each AMP. We show the antibacterial mode of action of AMPs can be accurately predicted by comparing ontological profiles generated by transcriptomic analyses. The response of E. coli to AMP challenge is highly plastic, with the bacteria capable of deploying a multifaceted response adapted to the mode of action rather than the physical properties of the AMP.
Project description:BACKGROUND: Defensins are a well known family of cationic antibacterial peptides (AMPs) isolated from fungi, plants, insects, mussels, birds, and various mammals. They are predominantly active against gram (+) bacteria, and a few of them are also active against gram (-) bacteria and fungi. All insect defensins belonging to the invertebrate class have a consensus motif, C-X₅₋₁₆-C-X₃-C-X₉₋₁₀-C-X₄₋₇-CX₁-C. Only seven AMPs have already been found in different lepidopteran species. No report was published on the isolation of defensin from the Egyptian cotton leafworm, Spodoptera littoralis. RESULTS: An anionic defensin, termed SpliDef, was isolated from the haemolymph of the cotton leafworm, S. littoralis, after bacterial challenge using differential display technique. Based on sequence analyses of the data, specific primers for full length and mature peptide of defensin were designed and successfully amplified 471 and 150 bp amplicons. The integration of the results revealed that the 471 bp-PCR product has one open reading frame (orf) of 303 bp long, including both start codon (AUG) and stop codon (UGA). The deduced peptide consists of a 23-residues signal peptide, a 27-residues propeptide and a 50-residues mature peptide with the conserved six-cysteine motif of insect defensins. Both haemolymph and expressed protein exhibited antibacterial activities comparable to positive control. The RT-qPCR indicated that it was more than 41-folds up-regulated at 48 h p.i. CONCLUSION: Our results highlight an important immune role of the defensin gene in Spodoptera littoralis by cooperating with other AMPs to control bacterial infection.
Project description:The interaction of antimicrobial peptides (AMPs) with the inner membrane of Gram-negative bacteria is a key determinant of their abilities to exert diverse bactericidal effects. Here we present a molecular level understanding of the initial target membrane interaction for two cationic ?-helical AMPs that share structural similarities but have a ten-fold difference in antibacterial potency towards Gram-negative bacteria. The binding and insertion from solution of pleurocidin or magainin 2 to membranes representing the inner membrane of Gram-negative bacteria, comprising a mixture of 128 anionic and 384 zwitterionic lipids, is monitored over 100?ns in all atom molecular dynamics simulations. The effects of the membrane interaction on both the peptide and lipid constituents are considered and compared with new and published experimental data obtained in the steady state. While both magainin 2 and pleurocidin are capable of disrupting bacterial membranes, the greater potency of pleurocidin is linked to its ability to penetrate within the bacterial cell. We show that pleurocidin displays much greater conformational flexibility when compared with magainin 2, resists self-association at the membrane surface and penetrates further into the hydrophobic core of the lipid bilayer. Conformational flexibility is therefore revealed as a key feature required of apparently ?-helical cationic AMPs for enhanced antibacterial potency.
Project description:The transition of antimicrobial peptides (AMPs) from the laboratory to market has been severely hindered by their instability toward proteases in biological systems. In the present study, we synthesized derivatives of the cationic AMP Pep05 (KRLFKKLLKYLRKF) by substituting L-amino acid residues with D- and unnatural amino acids, such as D-lysine, D-arginine, L-2,4-diaminobutanoic acid (Dab), L-2,3-diaminopropionic acid (Dap), L-homoarginine, 4-aminobutanoic acid (Aib), and L-thienylalanine, and evaluated their antimicrobial activities, toxicities, and stabilities toward trypsin, plasma proteases, and secreted bacterial proteases. In addition to measuring changes in the concentration of the intact peptides, LC-MS was used to identify the degradation products of the modified AMPs in the presence of trypsin and plasma proteases to determine degradation pathways and examine whether the amino acid substitutions afforded improved proteolytic resistance. The results revealed that both D- and unnatural amino acids enhanced the stabilities of the peptides toward proteases. The derivative DP06, in which all of the L-lysine and L-arginine residues were replaced by D-amino acids, displayed remarkable stability and mild toxicity <i>in vitro</i> but only slight activity and severe toxicity <i>in vivo</i>, indicating a significant difference between the <i>in vivo</i> and <i>in vitro</i> results. Unexpectedly, we found that the incorporation of a single Aib residue at the N-terminus of compound UP09 afforded remarkably enhanced plasma stability and improved activity <i>in vivo</i>. Hence, this derivative may represent a candidate AMP for further optimization, providing a new strategy for the design of novel AMPs with improved bioavailability.
Project description:More than 40 antimicrobial peptides and proteins (AMPs) are expressed in the oral cavity. These AMPs have been organized into 6 functional groups, 1 of which, cationic AMPs, has received extensive attention in recent years for their promise as potential antibiotics. The goal of this review is to describe recent advances in our understanding of the diverse mechanisms of action of cationic AMPs and the bacterial resistance against these peptides. The recently developed peptide GL13K is used as an example to illustrate many of the discussed concepts. Cationic AMPs typically exhibit an amphipathic conformation, which allows increased interaction with negatively charged bacterial membranes. Peptides undergo changes in conformation and aggregation state in the presence of membranes; conversely, lipid conformation and packing can adapt to the presence of peptides. As a consequence, a single peptide can act through several mechanisms depending on the peptide's structure, the peptide:lipid ratio, and the properties of the lipid membrane. Accumulating evidence shows that in addition to acting at the cell membrane, AMPs may act on the cell wall, inhibit protein folding or enzyme activity, or act intracellularly. Therefore, once a peptide has reached the cell wall, cell membrane, or its internal target, the difference in mechanism of action on gram-negative and gram-positive bacteria may be less pronounced than formerly assumed. While AMPs should not cause widespread resistance due to their preferential attack on the cell membrane, in cases where specific protein targets are involved, the possibility exists for genetic mutations and bacterial resistance. Indeed, the potential clinical use of AMPs has raised the concern that resistance to therapeutic AMPs could be associated with resistance to endogenous host-defense peptides. Current evidence suggests that this is a rare event that can be overcome by subtle structural modifications of an AMP.
Project description:As bacterial resistance to traditional antibiotics continues to emerge, new alternatives are urgently needed. Antimicrobial peptides (AMPs) are important candidates. However, how AMPs are designed with in vivo efficacy is poorly understood. Our study was designed to understand structural moieties of cationic peptides that would lead to their successful use as antibacterial agents. In contrast to the common perception, serum binding and peptide stability were not the major reasons for in vivo failure in our studies. Rather, our systematic study of a series of peptides with varying lysines revealed the significance of low cationicity for systemic in vivo efficacy against Gram-positive pathogens. We propose that peptides with biased amino acid compositions are not favored to associate with multiple host factors and are more likely to show in vivo efficacy. Thus, our results uncover a useful design strategy for developing potent peptides against multidrug-resistant pathogens.
Project description:To combat the escalating rise of antibacterial resistance, the development of antimicrobial peptides (AMPs) with a unique mode of action is considered an attractive strategy. However, proteolytic degradation of AMPs remains the greatest challenge in their transformation into therapeutics. Herein, we synthesized Fmoc-triazine amino acids that differ from each other by anchoring either cationic or hydrophobic residues. These unnatural amino acids were adopted for solid-phase peptide synthesis (SPPS) to synthesize a series of amphipathic antimicrobial peptidomimetics. From the antimicrobial screening, we found that the trimer, BJK-4 is the most potent short antimicrobial peptidomimetic without showing hemolytic activity and it displayed enhanced proteolytic stability. Moreover, the mechanism of action to kill bacteria was found to be an intracellular targeting.
Project description:Antimicrobial peptides (AMPs) offer great hope and a promising opportunity to overcome the rapid development of drug-resistant pathogenic microbes. However, AMPs often lack the stability required for a successful systemic drug. Hybridizing different AMPs is a simple and effective strategy to obtain novel peptides. N-terminal fragment of cecropin A (CA (1-8)) is often used to hybridize with other AMPs to reduce cytotoxicity. However, hybridizing with CA (1-8) in improving the stability of AMPs is not clear. Therefore, a series of peptides were designed by combining with CA (1-8) and their antibacterial activity and stability in the presence of salts and human serum were evaluated. The resultant ?-helical hybrid peptide CA-FO composed of CA (1-8) and the most potent region of Fowlicidin-2 (FO (1-15)) exhibited excellent antibacterial activity (2-8 ?M) and cell selectivity toward bacterial over mammalian cells. Moreover, CA-FO still retained vigorous antimicrobial activity in the presence of human serum and salts at physiological concentrations. CA-FO exhibited effective antibacterial activity by increasing membrane permeability and damaging membrane integrity. In conclusion, these results indicated the success of hybridization in designing and optimizing AMPs with improved stability and selectivity and the peptide CA-FO can be further evaluated as peptide-therapy to treat bacterial infections.
Project description:Cationic antimicrobial peptides (AMPs) are active immune effectors of multicellular organisms and are also considered as new antimicrobial drug candidates. One of the problems encountered when developing AMPs as drugs is the difficulty of reaching sufficient killing concentrations under physiological conditions. Here, using pexiganan, a cationic peptide derived from a host defense peptide of the African clawed frog and the first AMP developed into an antibacterial drug, we studied whether sub-lethal effects of AMPs can be harnessed to devise treatment combinations. We studied the pexiganan stress response of Staphylococcus aureus at sub-lethal concentrations using quantitative proteomics. Several proteins involved in nucleotide metabolism were elevated, suggesting a metabolic demand. We then show that Staphylococcus aureus is highly susceptible to antimetabolite nucleoside analogs when exposed to pexiganan, even at sub-inhibitory concentrations. These findings could be used to enhance pexiganan potency while decreasing the risk of resistance emergence, and our findings can likely be extended to other antimicrobial peptides.