A Novel Dimeric Short Peptide Derived from α-Defensin-Related Rattusin with Improved Antimicrobial and DNA-Binding Activities.

Rattusin, an α-defensin-related antimicrobial peptide isolated from the small intestine of rats, has been previously characterized through NMR spectroscopy to elucidate its three-dimensional structure, revealing a C2 homodimeric scaffold stabilized by five disulfide bonds. This study aimed to identify the functional region of rattusin by designing and synthesizing various short analogs, subsequently leading to the development of novel peptide-based antibiotics. The analogs, designated as F1, F2, F3, and F4, were constructed based on the three-dimensional configuration of rattusin, among which F2 is the shortest peptide and exhibited superior antimicrobial efficacy compared to the wild-type peptide. The central cysteine residue of F2 prompted an investigation into its potential to form a dimer at neutral pH, which is critical for its antimicrobial function. This activity was abolished upon the substitution of the cysteine residue with serine, indicating the necessity of dimerization for antimicrobial action. Further, we synthesized ß-hairpin-like analogs, both parallel and antiparallel, based on the dimeric structure of F2, which maintained comparable antimicrobial potency. In contrast to rattusin, which acts by disrupting bacterial membranes, the F2 dimer binds directly to DNA, as evidenced by fluorescence assays and DNA retardation experiments. Importantly, F2 exhibited negligible cytotoxicity up to 515 µg/mL, assessed via hemolysis and MTT assays, underscoring its potential as a lead compound for novel peptide-based antibiotic development.

The Function and Modification of Human Defensin 5.

The antibacterial and antiviral functions of human defensin 5 lay the foundation for its role as a core host protective component. In addition, HD5 also has the function of inhibiting tumor proliferation and immune regulation. However, everything has two sides; cytotoxic and proinflammatory properties may exist, while HD5 performs physiological functions. Accordingly, the modification and engineering of HD5 are particularly important. Therefore, this review summarizes the role of HD5 in various aspects of host defense, as well as modification of HD5 to ameliorate the biological activity, with a view to promoting the clinical use of HD5.

Efficient recombinant production of mouse-derived cryptdin family peptides by a novel facilitation strategy for inclusion body formation.

BACKGROUND: A number of antimicrobial peptides (AMPs) hold promise as new drugs owing to their potent bactericidal activity and because they are often refractory to the development of drug resistance. Cryptdins (Crps) are a family of antimicrobial peptides found in the small intestine of mice, comprising six isoforms containing three sets of disulfide bonds. Although Crp4 is actively being investigated, there have been few studies to date on the other Crp isoforms. A prerequisite for detailed characterization of the other Crp isoforms is establishment of efficient sample preparation methods. RESULTS: To avoid degradation during recombinant expression of Crps in E. coli, co-expression of Crps with the aggregation-prone protein human α-lactalbumin (HLA) was used to promote the formation of stable inclusion bodies. Using this method, the production of Crp4 and Crp6 by the BL21 strain was effective, but the expression of other Crp isoforms was not as efficient. The results of a cell-free system study suggested that Crps were degraded, even though a substantial amounts of Crps were synthesized. Therefore, using the Origami™ B strain, we were able to significantly increase the expression efficiency of Crps by promoting the formation of erroneous intermolecular disulfide bonds between HLA and Crps, thereby promoting protein aggregation and inclusion body formation, which prevented degradation. The various Crp isoforms were successfully refolded in vitro and purified using reversed-phase HPLC. In addition, the yield was further improved by deformylation of formyl-Crps. We measured the antibacterial activity of Crps against both Gram-positive and Gram-negative bacteria. Each Crp isoform exhibited a completely different trend in antimicrobial activity, although conformational analysis by circular dichroism did not reveal any significant steric differences. CONCLUSION: In this study, we established a novel and efficient method for the production of the cryptdin family of cysteine-containing antimicrobial peptides. Additionally, we found that there were notable differences in the antibacterial activities of the various Crp family members. The expression system established in this study is expected to provide new insights regarding the mechanisms underlying the different antibacterial activities of the Crp family of peptides.

Exploring the impact of the recombinant Escherichia coli strain on defensins antimicrobial activity: BL21 versus Origami strain.

The growing emergence of microorganisms resistant to antibiotics has prompted the development of alternative antimicrobial therapies. Among them, the antimicrobial peptides produced by innate immunity, which are also known as host defense peptides (HDPs), hold great potential. They have been shown to exert activity against both Gram-positive and Gram-negative bacteria, including those resistant to antibiotics. These HDPs are classified into three categories: defensins, cathelicidins, and histatins. Traditionally, HDPs have been chemically synthesized, but this strategy often limits their application due to the high associated production costs. Alternatively, some HDPs have been recombinantly produced, but little is known about the impact of the bacterial strain in the recombinant product. This work aimed to assess the influence of the Escherichia coli strain used as cell factory to determine the activity and stability of recombinant defensins, which have 3 disulfide bonds. For that, an α-defensin [human α-defensin 5 (HD5)] and a ß-defensin [bovine lingual antimicrobial peptide (LAP)] were produced in two recombinant backgrounds. The first one was an E. coli BL21 strain, which has a reducing cytoplasm, whereas the second was an E. coli Origami B, that is a strain with a more oxidizing cytoplasm. The results showed that both HD5 and LAP, fused to Green Fluorescent Protein (GFP), were successfully produced in both BL21 and Origami B strains. However, differences were observed in the HDP production yield and bactericidal activity, especially for the HD5-based protein. The HD5 protein fused to GFP was not only produced at higher yields in the E. coli BL21 strain, but it also showed a higher quality and stability than that produced in the Origami B strain. Hence, this data showed that the strain had a clear impact on both HDPs quantity and quality.

Potent bactericidal activity of reduced cryptdin-4 derived from its hydrophobicity and mediated by bacterial membrane disruption.

Defensin is a cysteine-rich antimicrobial peptide with three disulphide bonds under normal oxidative conditions. Cryptdin-4 (Crp4) is a defensin secreted by Paneth cells in the small intestine of mice, and only reduced Crp4 (Crp4red) shows activity against enteric commensal bacteria, although both oxidised Crp4 (Crp4ox) and Crp4red can kill non-commensal bacteria. To investigate the molecular factors that affect the potent antimicrobial activity of Crp4red, the bactericidal activities of Crp4ox and Crp4red, Crp4 with all Cys residues substituted with Ser peptide (6C/S-Crp4), and Crp4 with all thiol groups modified by N-ethylmaleimide (NEM-Crp4) were assessed. All peptides showed bactericidal activity against non-commensal bacteria, whereas Crp4red and NEM-Crp4 showed bactericidal activity against commensal bacteria. These potent peptides exhibited high hydrophobicity, which was strongly correlated with membrane insertion. Intriguingly, Crp4ox formed electrostatic interactions with the membrane surface of bacteria, even without exerting bactericidal activity. Moreover, the bactericidal activity of both oxidised and reduced forms of Crp4 was abolished by inhibition of electrostatic interactions; this finding suggests that Crp4red targets bacterial membranes. Finally, a liposome leakage assay against lipids extracted from commensal bacteria demonstrated a correlation with bactericidal activity. These results suggest that the potent bactericidal activity of Crp4red is derived from its hydrophobicity, and the bactericidal mechanism involves disruption of the bacterial membrane. Findings from this study provide a better understanding of the bactericidal mechanism of both Crp4ox and Crp4red.

Computational evaluation of modified peptides from human neutrophil peptide 1 (HNP-1).

The development of bacterial resistance toward antibiotics has been led to pay attention to the antimicrobial peptides (AMPs). The common mechanism of AMPs is disrupting the integrity of the bacterial membrane. One of the most accessible targets for α-defensins human neutrophil peptide-1 (HNP-1) is lipid II. In the present study, we performed homology modeling and geometrical validation of human neutrophil defensin 1. Then, the conformational and physicochemical properties of HNP-1 derived peptides 2Abz14S29, 2Abz23S29, and HNP1ΔC18A, as well as their interaction with lipid II were studied computationally. The overall quality of the predicted model of full protein was -5.14, where over 90% of residues were in the most favored and allowed regions in the Ramachandran plot. Although HNP-1 and HNP1ΔC18A were classified as unstable peptides, 2Abz14S29 and 2Abz23S29 were stable, based on the instability index values. Molecular docking showed similar interaction pattern of peptides and HNP-1 to lipid II. Molecular dynamic simulations revealed the overall stability of conformations, though the fluctuations of amino acids in the modified peptides were relatively higher than HNP-1. Further, the binding affinity constant (Kd) of HNP-1 and 2Abz23S29 in complex with lipid II was 10 times stronger than 2Abz14S29 and HNP1ΔC18A. Overall, computational studies of conformational and interaction patterns have signified how derived peptides could have displayed relatively similar antimicrobial results compared to HNP-1 in the reported experimental studies. Chemical modifications not only have improved the physicochemical properties of derived peptides compared to HNP-1, but also they have retained the similar pattern and binding affinity of peptides. Communicated by Ramaswamy H. Sarma.

Evaluation of the Binding Mechanism of Human Defensin 5 in a Bacterial Membrane: A Simulation Study.

Human α-defensin 5 (HD5) is a host-defense peptide exhibiting broad-spectrum antimicrobial activity. The lipopolysaccharide (LPS) layer on the Gram-negative bacterial membrane acts as a barrier to HD5 insertion. Therefore, the pore formation and binding mechanism remain unclear. Here, the binding mechanisms at five positions along the bacterial membrane axis were investigated using Molecular Dynamics. (MD) simulations. We found that HD5 initially placed at positions 1 to 3 moved up to the surface, while HD5 positioned at 4 and 5 remained within the membrane interacting with the middle and inner leaflet of the membrane, respectively. The arginines were key components for tighter binding with 3-deoxy-d-manno-octulosonic acid (KDO), phosphates of the outer and inner leaflets. KDO appeared to retard the HD5 penetration.

The penetration of human defensin 5 (HD5) through bacterial outer membrane: simulation studies.

Human α-defensin 5 (HD5) is one of cationic antimicrobial peptides which plays a crucial role in an innate immune system in human body. HD5 shows the killing activity against a broad spectrum of pathogenic bacteria by making a pore in a bacterial membrane and penetrating into a cytosol. Nonetheless, its pore-forming mechanisms remain unclear. Thus, in this work, the constant-velocity steered molecular dynamics (SMD) simulation was used to simulate the permeation of a dimeric HD5 into a gram-negative lipopolysaccharide (LPS) membrane model. Arginine-rich HD5 is found to strongly interact with a LPS surface. Upon arrival, arginines on HD5 interact with lipid A head groups (a top part of LPS) and then drag these charged moieties down into a hydrophobic core resulting in the formation of water-filled pore. Although all arginines are found to interact with a membrane, Arg13 and Arg32 appear to play a dominant role in the HD5 adsorption on a gram-negative membrane. Furthermore, one chain of a dimeric HD5 is required for HD5 adhesion. The interactions of arginine-lipid A head groups play a major role in adhering a cationic HD5 on a membrane surface and retarding a HD5 passage in the meantime.

Defensin 5 for prevention of SARS-CoV-2 invasion and Covid-19 disease.

Corona virus disease 2019 (Covid-19), a pandemia emerged recently, caused by severe acute respiratory syndrome corona virus 2 (SARS-CoV-2). The receptor for corona virus and influenza A is the mucosal cell membrane protein angiotensin converting enzyme 2 (ACE2), which is abundant on the membrane of alveolar cells and enterocytes. Viral spike protein 1 (S1) is the ligand, with an affinity of 14.7 nM to the receptor. The main port of entry for the virus is the upper respiratory tract, and the diagnosis is usually by PCR of the viral RNA with nasal and pharyngeal swab test. Human defensin 5 (HDEF5) is a protein encoded by the DEFA gene, secreted by Paneth cells in the small intestine and by granules of neutrophils. It has an affinity of 39.3 nM to ACE2, much higher than that of the corona S1. HDEF5 may also attach to glycosylated Corona S1 protein, make its efficiency even better. The issues to be investigated are the affinity of HDEF5 to S1 protein, the ability of recombinant HDEF5 function in attaching both ACE2 and S1, and the feasibility to perform aerosol spray of this protein. In addition, safety and efficiency should be studied in phases I, II and II clinical protocols. Thus, an aerosol spray of HDEF5 given through the nose and throat, once to several times a day, may be a very efficient approach to prevent infection with SARA-CoV-2 as well as influenza A.

Paneth cell α-defensin misfolding correlates with dysbiosis and ileitis in Crohn's disease model mice.

Crohn's disease (CD) is an intractable inflammatory bowel disease, and dysbiosis, disruption of the intestinal microbiota, is associated with CD pathophysiology. ER stress, disruption of ER homeostasis in Paneth cells of the small intestine, and α-defensin misfolding have been reported in CD patients. Because α-defensins regulate the composition of the intestinal microbiota, their misfolding may cause dysbiosis. However, whether ER stress, α-defensin misfolding, and dysbiosis contribute to the pathophysiology of CD remains unknown. Here, we show that abnormal Paneth cells with markers of ER stress appear in SAMP1/YitFc, a mouse model of CD, along with disease progression. Those mice secrete reduced-form α-defensins that lack disulfide bonds into the intestinal lumen, a condition not found in normal mice, and reduced-form α-defensins correlate with dysbiosis during disease progression. Moreover, administration of reduced-form α-defensins to wild-type mice induces the dysbiosis. These data provide novel insights into CD pathogenesis induced by dysbiosis resulting from Paneth cell α-defensin misfolding and they suggest further that Paneth cells may be potential therapeutic targets.

Bacterial Membrane Selective Antimicrobial Peptide-Mimetic Polyurethanes: Structure-Property Correlations and Mechanisms of Action.

The rise in prevalence of antibiotic resistant strains of bacteria is a very significant challenge for treating life-threatening infections worldwide. A source of novel therapeutics that has shown great promise is a class of biomolecules known as antimicrobial peptides. Previously, within our laboratories, we developed a new family of water-soluble antimicrobial polyurethanes that mimic antimicrobial peptides. Within this current investigation, studies were carried out to gain a greater understanding of the structure/property relationships of the polyurethanes. This was achieved by synthesizing a variety of pendant group functionalized polyurethanes and testing their effectiveness as an antimicrobial by carrying out minimum inhibitory concentration testing and determining their compatibility with blood cells. Additionally, insight into the mode of action of the polyurethanes was obtained through experiments using dye encapsulated phospholipids and assays of bacterial cells that indicated the ability of the polyurethanes to penetrate and disrupt membranes. Collectively, the results indicate that the addition of hydrophobic, uncharged polar, and anionic moieties do not have a strong influence on the antimicrobial activity; yet, the addition of hydrophobic groups enhances cytoplasmic membrane disruption, a larger proportion of cationic pendant groups promotes greater outer membrane disruption of Gram negative bacteria, and uncharged polar groups and anionic groups improve compatibility of the polyurethanes with mammalian cells.

Antimicrobial peptide ROAD-1 triggers phase change in local membrane environment to execute its activity.

Emergence of antibiotic-resistant pathogens has paved way for development of newer class of drugs that would not be susceptible to resistance. Antimicrobial peptides such as defensins that target the microbial membrane are promising candidates. ROAD-1 is an alpha-defensin present in the oral cavity of rhesus macaque and shares very high sequence similarity to human enteric defensin 5. In this study we have performed microsecond long all atom molecular dynamic simulations to understand the mechanism of action of ROAD-1. We find that ROAD-1 is able to adopt an energetically stable conformation predominantly stabilized by electrostatic interactions only in presence of bacterial membranes. In mammalian membrane even though it gets absorbed onto the bilayer, it is unable to adopt an equilibrium conformation. Binding of ROAD-1 to bilayer induces clustering of POPG molecules up to 15 Å around the peptide. POPG molecules show higher order parameters than the neighboring POPE implying coexistence of different phases. Analysis of binding free energy of ROAD-1-membrane complex indicates Arg1, Arg2, Arg7, and Arg25 to play key role in its antimicrobial activity. Unlike its homolog HD5, ROAD-1 is not observed to form a dimer. Our study gives insight into the membrane-bound conformation of ROAD-1 and its mechanism of action that can aid in designing defensin-based therapeutics. Graphical abstract Antimicrobial peptide ROAD-1 adopts a different membrane-bound conformation as compared with HD5 even though they belong to the same family implying a different mechanism of action.

Dynamics of human defensin 5 (HD5) self-assembly in solution: Molecular simulations/insights.

Human α -defensin 5 (HD5) is a 32-residue cysteine-rich host-defense peptide that exhibits broad-spectrum antimicrobial activity and plays an essential role in innate immunity in the human gut and other organ systems. Although its antimicrobial mechanism of action remains unclear, the high salt concentration seems to attenuate the antimicrobial function of HD5 via an unknown mechanism. In this work, we employ Molecular Dynamics (MD) simulations to analyse the oligomerization behaviour of HD5 when exposed to different salt concentration. We demonstrate that the presence of salt, such as sodium chloride (NaCl), promotes HD5 to form higher-order oligomers (up to heptamers) in our simulations. In addition, we also analyse the electrostatic interactions between the two Glu residues (E14 and E21) and their neighbouring residues. Our data confirm that the E14 residue is essential for the structural integrity, whereas the E21 residue contributes to the dimerization of HD5, suggesting that these Glu residues are important for the antimicrobial function of this peptide.

Human α defensins promote the expression of the inflammatory cytokine interleukin-8 under high-glucose conditions: Novel insights into the poor healing of diabetic foot ulcers.

Sustained infection and chronic inflammation are the most common features and complex mechanisms of diabetic foot disease. In this study, we examined the expression and functional roles of human endogenous α defensins in diabetic foot ulcer. The expression levels of human α defensins HNP1, HNP3, and HNP4 were significantly higher in the wound center than the edge of diabetic foot ulcers. And the inflammatory cytokine interleukin IL-8 (IL-8) was also highly expressed in wound exudates. In human foreskin fibroblasts, these human α defensins were found only slightly to affect IL-8 expression directly. hemoglobin A1C (HbA1c) is the main clinical indicator of diabetic foot disease. Advanced glycation end products of bovine serum albumin (AGE-BSA), as HbA1c analogue, was found to promote IL-8 expression. Human α defensins, in the presence of AGE-BSA, further significantly promoted IL-8 expression. These findings showed that human α defensins aggravated the inflammatory response in diabetic foot ulcers patients, providing new insights in to the poor healing of diabetic foot ulcers.

Critical determinants of human neutrophil peptide 1 for enhancing host epithelial adhesion of Shigella flexneri.

Human neutrophil peptides (HNPs), also known as human myeloid α-defensins degranulated by infiltrating neutrophils at bacterial infection loci, exhibit broad antomicrobial activities against bacteria, fungi, and viruses. We have made a surprising recent finding that Shigella, a highly contagious, yet poorly adhesive enteric pathogen, exploits human α-defensins including HNP1 to enhance its adhesion to and invasion of host epithelial cells. However, the critical molecular determinants responsible for HNP1-enhanced Shigella adhesion and invasion have yet to be investigated. Using cultured epithelial cells and polarised Caco2 cells as an in vitro infection model, we demonstrated that HNP1 promoted Shigella infection in a structure- and sequence-dependent manner, with two bulky hydrophobic residues, Trp26 and Phe28 important for HNP1 self-assembly, being most critical. The functional importance of hydrophobicity for HNP1-enhanced Shigella infection was further verified by substitutions for Trp26 of a series of unnatural amino acids with straight aliphatic side chains of different lengths. Dissection of the Shigella infection process revealed that bacteria-rather than host cells-bound HNP1 contributed most to the enhancement. Further, mutagenesis analysis of bacterial surface components, while precluding the involvement of lipopolysaccharides (LPS) in the interaction with HNP1, identified outer membrane proteins and the Type 3 secretion apparatus as putative binding targets of HNP1 involved in enhanced Shigella adhesion and invasion. Our findings provide molecular and mechanistic insights into the mode of action of HNP1 in promoting Shigella infection, thus showcasing another example of how innate immune factors may serve as a double-edged sword in health and disease.

Exploiting chitosan and gold nanoparticles for antimycobacterial activity of in silico identified antimicrobial motif of human neutrophil peptide-1.

The upsurge of drug resistant tuberculosis is major health threat globally. To counteract, antimicrobial peptides are being explored as possible alternatives. However, certain limitations of peptide-based drugs such as potential toxicity, high cost and relatively low stability need to be addressed to enhance their clinical applicability. Use of computer predicted short active motifs of AMPs along with nanotechnology could not only overcome the limitations of AMPs but also potentiate their antimicrobial activity. Therefore, present study was proposed to in silico identify short antimicrobial motif (Pep-H) of human neutrophil peptide-1 (HNP-1) and explore its antimycobacterial activity in free form and using nanoparticles-based delivery systems. Based on colony forming unit analysis, motif Pep-H led to killing of more than 90% M. tb in vitro at 10 µg/ml, whereas, similar activity against intracellularly growing M. tb was observed at 5 µg/ml only. Thereafter, chitosan (244 nm) and gold nanoparticles (20 nm) were prepared for Pep-H with both the formulations showing minimal effects on the viability of human monocyte derived macrophages (MDMs) and RBC integrity. The antimycobacterial activity of Pep-H against intracellular mycobacteria was enhanced in both the nanoformulations as evident by significant reduction in CFU (>90%) at 5-10 times lower concentrations than that observed for free Pep-H. Thus, Pep-H is an effective antimycobacterial motif of HNP-1 and its activity is further enhanced by chitosan and gold nanoformulations.

Newly Identified HNP-F from Human Neutrophil Peptide-1 Promotes Hemostasis.

Active hemostatic agents can play a crucial role in saving patients' lives during surgery. Active hemostats have several advantages including utilization of natural blood coagulation and biocompatibility. Among them, although human neutrophil peptide-1 (HNP-1) has been previously reported with the hemostatic mechanism, which part of HNP-1 facilitates the hemostatic activity is not known. Here, a partial peptide (HNP-F) promoting hemostasis, originating from HNP-1, has been newly identified by the blood coagulation ability test. HNP-F shows the best hemostatic effect between the anterior half and posterior half of peptides. Moreover, microscopic images show platelet aggregation and an increase in the concentration of platelet factor 4, and the scanning electron microscope image of platelets support platelet activation by HNP-F. Thromboelastography indicates decreased clotting time and increased physical properties of blood clotting. Mouse liver experiments demonstrate improved hemostatic effect by treatment of peptide solution. Cell viability and hemolysis assays confirm the HNP-F's biosafety. It is hypothesized that the surface charge and structure of HNP-F could be favorable to interact with fibrinogen or thrombospondin-1. Collectively, because HNP-F as an active peptide hemostat has many advantages, it could be expected to become a potent hemostatic biomaterial, additive or pharmaceutical candidate for various hemostatic applications.

Systematic mutational analysis of human neutrophil α-defensin HNP4.

Defensins are a family of cationic antimicrobial peptides of innate immunity with immunomodulatory properties. The prototypic human α-defensins, also known as human neutrophil peptides 1-3 or HNP1-3, are extensively studied for their structure, function and mechanisms of action, yet little is known about HNP4 - the much less abundant "distant cousin" of HNP1-3. Here we report a systematic mutational analysis of HNP4 with respect to its antibacterial activity against E. coli and S. aureus, inhibitory activity against anthrax lethal factor (LF), and binding activity for LF and HIV-1 gp120. Except for nine conserved and structurally important residues (6xCys, 1xArg, 1xGlu and 1xGly), the remaining 24 residues of HNP4 were each individually mutated to Ala. The crystal structures of G23A-HNP4 and T27A-HNP4 were determined, both exhibiting a disulfide-stabilized canonical α-defensin dimer identical to wild-type HNP4. Unlike HNP1-3, HNP4 preferentially killed the Gram-negative bacterium, a property largely attributable to three clustered cationic residues Arg10, Arg11 and Arg15. The cationic cluster was also important for HNP4 killing of S. aureus, inhibition of LF and binding to LF and gp120. However, F26A, while functionally inconsequential for E. coli killing, was far more deleterious than any other mutations. Similarly, N-methylation of Leu20 to destabilize the HNP4 dimer had little effect on E. coli killing, but significantly reduced the ability of HNP4 to kill S. aureus, inhibit LF, and bind to LF and gp120. Our findings unveil the molecular determinants of HNP4 function, completing the atlas of structure and function relationships for all human neutrophil α-defensins.

The adsorption of human defensin 5 on bacterial membranes: simulation studies.

Human α-defensin 5 (HD5) is one of the important antimicrobial peptides (AMPs) used against a broad-spectrum of pathogens, especially Gram-negative bacteria. HD5 kills by disrupting and making a pore in the bacterial membrane. The presence of lipopolysaccharide (LPS), located on a membrane surface, is found to have an impact on HD5's activity, where such binding mechanism in microscopic detail remains unclear. In this work, we therefore employed molecular dynamics (MD) simulations to investigate the binding mechanisms of HD5 on LPS in comparison to a bare DMPC lipid membrane. Two oligomers, dimer and tetramer, are studied here. Apparently, the membrane structure influences the protein binding affinity. HD5 binds tighter to a lipid membrane than LPS. Both dimeric and tetrameric HD5 can penetrate deeply into a phosphate layer in a lipid membrane, whereas only facial contacts are observed for LPS systems. The proteins appear to stay in the polar area instead of diving into a hydrophobic region. Furthermore, it happens in all cases that residues in the active region (A1, T2, R6, R13, R32) contribute to the membrane adsorption. The breakdown of tetramer into two dimers is also found. This implies that the dimer is more favorable for membrane binding. Moreover, both dimeric and tetrameric HD5 can significantly disrupt a LPS layer, whilst no serious distortion of lipid membrane is obtained. This emphasizes the importance of LPS on HD5 activity.

Antimicrobial Activity of Chimera Peptides Composed of Human Neutrophil Peptide 1 (HNP-1) Truncated Analogues and Bovine Lactoferrampin.

Three chimera peptides composed of bovine lactoferrampin and the analogue of truncated human neutrophil peptide 1 were synthesized by the solid-phase method. In two compounds peptide chains were connected via isopeptide bond, whereas in the third one disulfide bridge served as a linker. All three chimeras displayed significantly higher antimicrobial activity than the constituent peptides as well as their equimolar mixtures. The one with a disulfide bridge displayed selectivity toward Gram-positive bacteria and was able to penetrate bacterial cells. The chimeric peptides demonstrated low in vitro mammalian cytotoxicity, especially against benign cells. The significance of linker type was also reflected in the secondary structure and proteolytic stability of studied compounds. Presented results proved that such chimeras are good lead structures for designing antimicrobial drugs.