Histone-derived Antimicrobial Peptides Research Articles

Hybrids of Membrane-Translocating Antimicrobial Peptides Show Enhanced Activity through Membrane Permeabilization.

Antimicrobial peptides (AMPs) hold promise as useful tools to combat bacterial infection. Hybrid peptides, made by linking two independent AMPs together through peptide bonds, have the potential for enhancing antimicrobial activity. Here we explore hybrids created by combining two histone-derived antimicrobial peptides (HDAPs), BF2 and DesHDAP1, that each translocate across bacterial membranes. Our work represents the first systematic approach considering the activity and mechanism of hybrids made from two translocating AMPs. BF2/DesHDAP1 hybrids showed increased antimicrobial activity against both Gram-positive and Gram-negative bacteria compared with the parent peptides and no cytotoxicity against eukaryotic cells. Introducing amino acid linkers between the parent peptides did not further enhance the antibacterial activity. The increased antimicrobial activity comes from a mechanistic shift, as hybrid peptides show decreased translocation across bacterial cell membranes but increased membrane permeabilization compared to BF2 and DesHDAP1. These observations lay the groundwork for the further design of hybrid AMPs made from translocating peptides.

Histone derived antimicrobial peptides identified from Mytilus coruscus serum by peptidomics

Histones and their N-terminal or C-terminal derived peptides have been studied in vertebrates and presented as potential antimicrobial agents playing important roles in the innate immune defenses. Although histones and their derived peptides had been reported as components of innate immunity in invertebrates, the knowledge about the histone derived antimicrobial peptides (HDAPs) in invertebrates are still limited. Using a peptidomic technique, a set of peptide fragments derived from the histones was identified in this study from the serum of microbes challenged Mytilus coruscus. Among the 85 identified histone-derived-peptides with high confidence, 5 HDAPs were chemically synthesized and the antimicrobial activities were verified, showing strong growth inhibition against Gram-positive bacteria, Gram-negative bacteria, and fungus. The gene expression level of the precursor histones matched by representative HDAPs were further tested using q-PCR, and the results showed a significant upregulation of the histone gene expression levels in hemocytes, gill, and mantle of the mussel after immune stress. In addition, three identified HDAPs were selected for preparation of specific antibodies, and the corresponding histones and their derived C-terminal fragments were detected by Western blotting in the blood cell and serum of immune challenged mussel, respectively, indicating the existence of HDAPs in M. coruscus. Our findings revealed the immune function of histones in Mytilus, and confirmed the existence of HDAPs in the mussel. The identified Mytilus HDAPs represent a new source of immune effector with antimicrobial function in the innate immune system, and thus provide promising candidates for the treatment of microbial infections in aquaculture and medicine.

Members of the histone-derived antimicrobial peptide family from the pearl oyster Pinctada fucata martensii: Inhibition of bacterial growth

Because it is difficult to isolate standard antimicrobial peptides (AMPs) using traditional biochemical approaches, we designed, synthesized, and evaluated a series of structurally altered histone-derived AMPs (HDAPs) from the pearl oyster Pinctada fucata martensii using molecular cloning approaches. Four histone-homolog genes (PmH2A, PmH2B, PmH3, and PmH4-1) were identified, of which PmH2A and PmH2B had yet to be described. PmH2A and PmH2B were therefore cloned using Rapid Amplification of cDNA Ends (RACE) and characterized. Constitutive PmH2A and PmH2B mRNA expression was detected in all six pearl oyster tissues tested, with comparatively greater transcript abundance in the gonads. Because α-helical content, hydrophilicity index, and the presence of a proline hinge may be the three important factors influencing the antimicrobial efficacy of HDAPs, we synthesized a series of eight N- and C-terminally truncated or amino acid-substituted synthetic candidate HDAP analogs derived from PmH2A, PmH2B, PmH3, and PmH4-1. Only the PmH2A- and PmH4-derived AMPs inhibited bacterial growth. The PmH2A-derived AMPs were α-helical proteins, while the PmH4-derived AMPs were extended strand/random coil proteins. Our results suggested that having an α-helical structure was particularly important for the antibacterial efficacy of the PmH2A-derived peptides; amphipathic structures (hydrophilic index, 0.3 to −0.3) may enhance the antimicrobial function of both the PmH2A- and PmH4-derived peptides. The high antibacterial efficacy of one of the HDAP analogs studied, PmH2A-AMP (5–13) [KLLK]3, indicated that this protein may represent a promising candidate for the treatment of bacterial infections in aquaculture mollusk species. This first study of HDAPs from the pearl oyster P. f. martensii provides new insights into the design and function of highly effective antimicrobial peptides.

Measuring Cytotoxicity of the Designed Antimicrobial Peptides DesHDAP1 and DesHDAP1‐C on Human Embryonic Kidney Cells

Antimicrobial peptides (AMPs) have emerged as a promising alternative to traditional antibiotics, particularly in light of multi‐antibiotic resistant strains of bacteria. Histone derived AMPs (HDAPs) are an integral part of this toolset, with multiple mechanisms of action, strong antimicrobial effects, and protein design potential. Our lab has previously designed two robust antimicrobial peptides: DesHDAP1 and the subsequent DesHDAP1‐C, which has an additional cysteine residue to facilitate conjugation to other molecules for fluorescence imaging or attachment to active moieties. Previous work demonstrated that the cysteine mutation did not alter antibacterial activity of the DesHDAP1 peptide. However, it is also critical to determine whether this change affects potential toxicity against eukaryotic cells. Using dye‐based assays, we quantified cellular metabolism of human embryonic kidney cells as a proxy for cell viability. By optimizing this process and the data analysis, I was able to compare cytotoxicity of the two designed peptides to that of conventional antibiotics. This approach can be applied to other designed peptides, providing a blueprint for future toxicity studies on eukaryotic cells.

Investigating Potential Therapeutic Activity of Designed Histone Derived Antimicrobial Peptides through Hybridization and Antibiotic Cocktails

Modern medicine is contingent on innovation in order to best face the myriad of challenges to human health and the evolution of organisms such as bacteria. Due to increasing antibiotic resistance to small molecule antibiotics in bacteria, there is a need for alternative therapeutic routes and options. One potential alternative is antimicrobial peptides (AMPs), which have a broad range of antimicrobial activity and are found as part of the innate immune response of many organisms. Through research and exploration of these AMPs we look to improve their antimicrobial activity. Our current research investigates these two approaches utilizing novel histone derived antimicrobial peptides (HDAPs). These peptides are based on buforin II (BF2), DesHDAP1, and DesHDAP2. In one approach we hybridize two peptides into a single, longer AMP. In the other approach we combine AMPs with conventional antibiotics. Studies have shown that some hybrid peptides are more effective against bacteria and less toxic to mammalian cells than the lone parent peptides. Similarly, using two therapeutics can yield synergistic effects, proving more beneficial combined than alone. Thus far, the hybrid peptides demonstrate increased antibacterial activity compared to the individual parent peptides, and some show much greater activity depending on the order of the parent peptides and the amino acid linker utilized. DesHDAP2 has antimicrobial activity when used alone comparable to that of BF2. Trials running DesHDAP2 in combination with several conventional antibiotics with differing mechanisms of action have noted potentially synergistic combinations. Ongoing work will determine if there are any harmful side effects to eukaryotic cells from these two therapeutic approaches.

Investigating How Proline Linkers Enhance Hybrid Antimicrobial Peptide Activity

To combat increasing antibiotic resistance in bacteria, antimicrobial peptides (AMPs) have been proposed as alternatives to conventional antibiotic treatments. AMPs are naturally found in many organisms, including humans, as part of the innate immune response. One approach to designing novel AMPs is to form a hybrid AMP by connecting two individual AMPs, often with a linker consisting of one or more amino acids. Previous studies have shown that some hybrid AMPs have increased antibacterial activity and reduced cytotoxicity to eukaryotic cells. The amino acid linker and hybrid design used may impact antibacterial activity; however, these effects have not yet been systematically evaluated. This study examines hybrid AMPs formed from two translocating histone-derived antimicrobial peptides (HDAPs): the naturally occurring buforin II (BF2) and the synthesized peptide DesHDAP1. Previous work in our laboratory has shown that proline linkers enhance the activity of these BF2/DesHDAP1 hybrids. To investigate which structural feature of proline contributes to this observed increase in activity, additional BF2/DesHDAP1 hybrids were designed using glycine and N-methyl alanine linkers. Antibacterial activity and circular dichroism spectroscopy results suggest that proline's inability to hydrogen bond contributes more to antibacterial activity than physical rigidity. Molecular dynamics simulations were also performed to compare the hybrids’ structure and interactions with membranes on a molecular level. Ongoing work is investigating the activity of these BF2/DesHDAP1 hybrid AMPs against eukaryotic cells.

Synergy between Histone-Derived Antimicrobial Peptides and Conventional Antibiotics

The rapid emergence of multidrug-resistant bacteria in recent years has been a significant medical concern, rendering conventional antibiotics ineffective and necessitating the development of new drugs and therapeutic strategies. This study investigated the potential synergistic effects of a series of histone-derived antimicrobial peptides (HDAPs) and antibiotics on selected gram-positive and gram-negative bacterial strains: Escherichia coli, Acinetobacter baylyi, Enterobacter aerogenes, Staphylococcus epidermidis, Staphylococcus aureus, and Bacillus subtilis. Two membrane-translocating HDAPs, Buforin II (BFII) and DesHDAP1, and two membrane-permeabilizing HDAPs, DesHDAP2 and DesHDAP3A, were examined along with several conventional antibiotics that span a range of activities: 1) agents that inhibit the synthesis of the bacterial cell wall (ampicillin, cephalexin); 2) those that function through inhibiting nucleic acid replication (levofloxacin, rifampicin) and 3) those that function by inhibiting translation (kanamycin, tetracycline). We chose these antimicrobial agents to represent different mechanisms of action in order to assess the effects of these differences on synergy between agents. After determining the relative antimicrobial efficacies and minimum inhibitory concentrations (MICs) of each agent using microdilution and radial diffusion assays respectively, checkerboard assays were designed and performed to determine their combinatory effects in vitro. Time-dependent growth curves were also collected for each strain under each combination. When combined at subinhibitory concentrations, membrane-translocating HDAPs and cell wall-inhibiting antibiotics were generally synergistic. Membrane-translocating HDAPs exhibited mixed results of synergy and additive killing effects with cell wall-inhibiting antibiotics. Interestingly, antagonistic effects were observed for most cell translation-inhibiting antibiotics with all peptides, particularly for combinations involving kanamycin. with these particular results, further investigation and additional studies with a broader range of combinations should be conducted to better understand these patterns in different AMP-antibiotic combinations and identify synergy in specific combinations.

A histone H2A-derived antimicrobial peptide, Hipposin from mangrove whip ray, Himanturawalga: Molecular and functional characterisation.

Antimicrobial peptides (AMPs) are biologically dynamic molecules produced by all type of organisms as a fundamental component of their innate immune system. The present study deals with the identification of a histone H2A-derived antimicrobial peptide, Hipposin from mangrove whip ray, Himanturawalga. A 243 base pair fragment encoding 81 amino acid residues amplified from complementary DNA was identified as Hipposin and termed as Hw-Hip. Homologous analysis showed that Hw-Hip belongs to the Histone H2A superfamily and shares sequence identity with other histone-derived AMPs from fishes. Phylogenetic analysis of Hw-Hip displayed clustering with the fish H2A histones. Secondary structure analysis showed the presence of three α-helices and four random coils with a prominent proline hinge. The physicochemical properties of Hw-Hip are in agreement with the properties of antimicrobial peptides. A 39-mer active peptide sequence was released by proteolytic cleavage in silico. Functional characterisation of active peptide in silico revealed antibacterial, anticancer and antibiofilm activities making Hw-Hip a promising candidate for further exploration.

Can Molecular Dynamics Simulations Predict the Effect of Truncating Histone-Derived Antimicrobial Peptides?

Can Molecular Dynamics Simulations Predict the Effect of Truncating Histone-Derived Antimicrobial Peptides?

Discovery of Novel Antimicrobial Peptides from Varanus komodoensis (Komodo Dragon) by Large-Scale Analyses and De-Novo-Assisted Sequencing Using Electron-Transfer Dissociation Mass Spectrometry

Komodo dragons are the largest living lizards and are the apex predators in their environs. They endure numerous strains of pathogenic bacteria in their saliva and recover from wounds inflicted by other dragons, reflecting the inherent robustness of their innate immune defense. We have employed a custom bioprospecting approach combining partial de novo peptide sequencing with transcriptome assembly to identify cationic antimicrobial peptides from Komodo dragon plasma. Through these analyses, we identified 48 novel potential cationic antimicrobial peptides. All but one of the identified peptides were derived from histone proteins. The antimicrobial effectiveness of eight of these peptides was evaluated against Pseudomonas aeruginosa (ATCC 9027) and Staphylococcus aureus (ATCC 25923), with seven peptides exhibiting antimicrobial activity against both microbes and one only showing significant potency against P. aeruginosa. This study demonstrates the power and promise of our bioprospecting approach to cationic antimicrobial peptide discovery, and it reveals the presence of a plethora of novel histone-derived antimicrobial peptides in the plasma of the Komodo dragon. These findings may have broader implications regarding the role that intact histones and histone-derived peptides play in defending the host from infection. Data are available via ProteomeXChange with identifier PXD005043.

Development of Cell-Wall Deficient Bacteria for the Characterization of Histone-Derived Antimicrobial Peptides through Confocal Microscopy

Development of Cell-Wall Deficient Bacteria for the Characterization of Histone-Derived Antimicrobial Peptides through Confocal Microscopy

Analysis and Prediction of some Histone-derived Antimicrobial Peptides from Toads Duttaphrynus melanostictus and Phyrinoidis asper

Antimicrobial peptides in skin secretions of toads is a promising methods to combat a wide spectrum of bacteria. Histone H2A is a type of DNA-binding protein that acts as a precursor for several antimicrobial peptides. In toads (family Bufonidae) buforin I and buforin II are examples of antimicrobial peptides that derived from histone H2A. This study investigated the genetic diversity and phylogenetic analysis and in silico prediction of antimicrobial peptides derived from histone H2A of Duttaphrynus melanostictus and Phyrinoidis asper , which were collected from Bogor Agricultural University’s campus area. A new set of primers (Buf_fwd and Buf_rev) were designed by using PrimerBLAST, to amplify 393 nucleotides of the histone H2A gene that codes 131 amino acids. Haplotype diversity of both species are very low. Phylogenetic analysis shows the sample D. melanostictus and P. asper are separated to each other in two different clades. Several short predicted peptides from histone H2A show a potential as an antimicrobial peptides based on in silico prediction. Psychochemical characteristics and 3D structure of potent antimicrobial peptides are described.

The Role of Arginine and Lysine in Histone Derived Antimicrobial Peptides

To combat the rapid increase of antibiotic resistant bacteria scientists have turned to antimicrobial peptides. Antimicrobial peptides (AMPs) are small proteins within the innate immune system with the ability to kill bacteria, fungi and viruses. Previous research has shown that a greater number of arginine amino acids enhance the antimicrobial activity of some AMPs. This study investigates the role of basic amino acids in three histone derived antimicrobial peptides (HDAPs): buforin II, parasin and DesHDAP1.

Using Molecular Dynamics Simulations to Characterize the Role Played by Basic Residues in Interactions of HDAPs and Bacterial Lipid Membranes

Antimicrobial peptides are small, cationic peptides that induce bacterial cell death via cell lysis or interactions with intracellular components. Currently, with growing bacterial resistance to existing antimicrobial drugs there is an urgent need to study the potentcy of antimicrobial peptides and find ways to improve their activity. Most AMPs include positively charged amino acids that allow binding to negatively-charged bacterial membranes. Previous studies have shown that an increased composition of arginine versus lysine enhances the activity of two histone-derived antimicrobial peptides (HDAPs), buforin II (BF2) and DesHDAP1. Variants of these peptides with all basic residues modified to arginine had increased antibacterial activity and membrane translocation but unchanged membrane permeabilization. In this study, we use molecular dynamics simulations to study how the arginine-modified peptides interact with model bacterial lipid membranes (1:3 POPG:POPE) differently than wild-type and lysine-modified peptides. Simulations were performed with one and four peptides for wild type BF2 (BF2wt), arginine-modified BF2 (BF2R), and lysine-modified BF2 (BF2K). The results of 100ns simulations of each system show that BF2R has increased interactions with the lipid membrane and structural stability compared to the other peptides. The analysis of hydrogen bonding per lipids supports that anionic POPG lipids interact more with the peptides than zwitterionic POPE lipids, further emphasizing the role of the basic residues. Analogous simulations considering the lipid interactions of DesHDAP1 and its arginine and lysine mutants are ongoing. Our findings elucidate mechanistic details of peptide-lipid interactions that will become helpful in the development of AMPs with improved activity.

Characterization of Nucleic Acid Binding by the Histone-Derived Antimicrobial Peptides Buforin II and DesHDAP1

Antimicrobial peptides (AMPs), which are found in numerous living organisms, are active against a wide range of bacteria and other pathogens, making them promising candidates for alternatives to conventional antibiotics. While many AMPs inhibit bacterial growth through membrane disruption, some AMPs, including buforin II and DesHDAP1, are hypothesized to kill bacteria by binding to intracellular nucleic acids after translocating across the bacterial cell membrane without significant membrane permeabilization. To understand this lesser known mechanism, the peptide-nucleic acid binding interactions of these systems were investigated using several experimental and computational methods. The peptide-nucleic acid binding for buforin II and DesHDAP1 were measured experimentally using a fluorescence intercalator displacement (FID) assay. In these experiments, having an increased composition of basic residues that are arginine versus lysine were shown to promote binding for variants of both buforin II and DesHDAP1. When binding was tested using different combinations of DNA sequences, neither buforin II nor DesHDAP1 significantly favored particular DNA sequences. To provide structural explanations for experimental results, molecular dynamics (MD) simulations, pKa calculations, electrostatics calculations via component analysis were used. Using these methods, particular arginine residues within each peptide were found to interact more favorably with DNA. Results from MD simulations also showed that nucleic acid-peptide binding was mostly due to interactions between the peptide and phosphate backbone of nucleic acids, providing an explanation for the lack of sequence specificity observed experimentally. These insights regarding nucleic acid binding of buforin II and DesHDAP1, paired with a deeper understanding of the peptides’ structures and membrane interactions, are necessary for development of novel pharmaceutical applications using AMPs.

Role of arginine and lysine in the antimicrobial mechanism of histone-derived antimicrobial peptides

Translocation of cell-penetrating peptides is often promoted by increased content of arginine or other guanidinium groups. However, relatively little research has considered the role of these functional groups on antimicrobial peptide activity. This study compared the activity of three histone-derived antimicrobial peptides—buforin II, DesHDAP1, and parasin—with variants that contain only lysine or arginine cationic residues. These peptides operate via different mechanisms as parasin causes membrane permeabilization while buforin II and DesHDAP1 translocate into bacteria. For all peptides, antibacterial activity increased with increased arginine content. Higher arginine content increased permeabilization for parasin while it improved translocation for buforin II and DesHDAP1. These observations provide insight into the relative importance of arginine and lysine in these antimicrobial peptides.

Effects of Cationic Residues and Base Sequence in Nucleic Acid Binding of Histone-Derived Antimicrobial Peptides

Antimicrobial peptides (AMPs), which are found in numerous living organisms, are active against a wide range of bacteria and other pathogens. While many AMPs inhibit bacterial growth through membrane disruption, certain AMPs, including buforin II and DesHDAP1, are hypothesized to kill bacteria by binding to intracellular nucleic acids after translocating across the bacterial cell membrane. To understand this lesser known mechanism, the nucleic acid-peptide binding interactions of these systems were investigated using both experimental and computational methods. The nucleic acid-peptide binding for buforin II and DesHDAP1 were measured experimentally using a fluorescence intercalator displacement (FID) assay. In these experiments, having an increased composition of basic residues that are arginine versus lysine were shown to promote binding for variants of both buforin II and DesHDAP1. When binding was tested using nucleic acid sequences, neither buforin II nor DesHDAP1 significantly favored particular DNA or RNA sequences. To provide structural explanations for these results, molecular dynamics (MD) simulations and electrostatics calculations were used. Through these computational analyses, particular arginine residues within each peptide were found to interact more favorably with nucleic acids. Results from MD simulations also showed that nucleic acid-peptide binding was mostly due to interactions between the peptide and phosphate backbone of nucleic acids. Since these phosphate groups are identical for any DNA or RNA sequence, the prevalence of those interactions explained the lack of sequence specificity observed experimentally. These insights regarding nucleic acid binding of buforin II and DesHDAP1, paired with a deeper understanding of the peptides’ structures and membrane interactions, are necessary to develop AMPs for novel pharmaceutical applications.

Molecular Characterization and Phylogenetic Analysis of a Histone-Derived Antimicrobial Peptide Teleostin from the Marine Teleost Fishes, Tachysurus jella and Cynoglossus semifasciatus.

Antimicrobial peptides (AMPs) are host defense peptides that are well conserved throughout the course of evolution. Histones are classical DNA-binding proteins, rich in cationic amino acids, and recently appreciated as precursors for various histone-derived AMPs. The present study deals with identification of the potential antimicrobial peptide sequence of teleostin from the histone H2A of marine teleost fishes, Cynoglossus semifasciatus and Tachysurus jella. A 245 bp amplicon coding for 81 amino acids was obtained from the cDNA transcripts of these fishes. The first 52 amino acids from the N terminal of the peptide were identical to previously characterized histone-derived antimicrobial peptides. Molecular and physicochemical characterizations of the sequence were found to be in agreement with previously reported histone H2A-derived AMPs, suggesting the possible role of histone H2A in innate defense mechanism in fishes.

Novel histone-derived antimicrobial peptides use different antimicrobial mechanisms

The increase in multidrug resistant bacteria has sparked an interest in the development of novel antibiotics. Antimicrobial peptides that operate by crossing the cell membrane may also have the potential to deliver drugs to intracellular targets. Buforin 2 (BF2) is an antimicrobial peptide that shares sequence identity with a fragment of histone subunit H2A and whose bactericidal mechanism depends on membrane translocation and DNA binding. Previously, novel histone-derived antimicrobial peptides (HDAPs) were designed based on properties of BF2, and DesHDAP1 and DesHDAP3 showed significant antibacterial activity. In this study, their DNA binding, permeabilization, and translocation abilities were assessed independently and compared to antibacterial activity to determine whether they share a mechanism with BF2. To investigate the importance of proline in determining the peptides’ mechanisms of action, proline to alanine mutants of the novel peptides were generated. DesHDAP1, which shows significant similarities to BF2 in terms of secondary structure, translocates effectively across lipid vesicle and bacterial membranes, while the DesHDAP1 proline mutant shows reduced translocation abilities and antimicrobial potency. In contrast, both DesHDAP3 and its proline mutant translocate poorly, though the DesHDAP3 proline mutant is more potent. Our findings suggest that a proline hinge can promote membrane translocation in some peptides, but that the extent of its effect on permeabilization depends on the peptide's amphipathic properties. Our results also highlight the different antimicrobial mechanisms exhibited by histone-derived peptides and suggest that histones may serve as a source of novel antimicrobial peptides with varied properties.

Characterizing the Antibacterial Mechanism of Three Novel Histone-Derived Antimicrobial Peptides

The rise in antibiotic resistant pathogens has sparked interest in antimicrobial peptides (AMPs), especially those that are active against a wide variety of bacterial strains. Cell penetrating AMPs are of particular importance for their potential as both drug delivery systems and antimicrobial agents. Buforin II (BF2) is a well-characterized antimicrobial peptide derived from histone subunit H2A that kills bacteria by translocating across cell membranes and binding to nucleic acids. In this study, we compared the mechanisms of action of three novel histone-derived antimicrobial peptides (HDAPs), termed DesHDAP1-3, to that of BF2. DesHDAP1's antibacterial potency is similar to that of BF2 across several bacterial species, while DesHDAP2 and DesHDAP3 are generally weaker antibacterial agents. Our current data also implies that DesHDAP1 shows increased cytotoxicity against cancerous cells lines compared to BF2. Lipid vesicle studies measuring the translocation of all three designed peptides showed that DesHDAP2 does not cross lipid membranes as readily as DesHDAP1, DesHDAP3, or BF2, which may explain its poor antibacterial activity. For comparison, we have also considered the membrane permeabilization caused by the designed peptides using a propidium iodide uptake assay. Finally, we have considered the role of nucleic acid binding in the mechanism of the designed peptides by measuring the DNA binding and antimicrobial activity of mutant versions of the designed peptides. This data shows that all three peptides may not show the same correlation between DNA binding and activity observed for BF2. An understanding of how the designed peptides function is an important step in assessing their therapeutic potential and considering future design strategies.