Translocation of Antimicrobial Peptides across Model Membranes: The Role of Peptide Chain Length.

Cushioned lipid bilayers are structures consisting of a lipid bilayer supported on a solid substrate with an intervening layer of soft material. They offer possibilities for studying the behavior and interactions of biological membranes more accurately under physiological conditions. In this work, we continue our studies of cushion formation induced by histatin 5 (24Hst5), focusing on the effect of the length of the peptide chain. 24Hst5 is a short, positively charged, intrinsically disordered saliva peptide, and here, both a shorter (14Hst5) and a longer (48Hst5) peptide variant were evaluated. Experimental surface active techniques were combined with coarse-grained Monte Carlo simulations to obtain information about these peptides. Results show that at 10 mM NaCl, both the shorter and the longer peptide variants behave like 24Hst5 and a cushion below the bilayer is formed. At 150 mM NaCl, however, no interaction is observed for 24Hst5. On the contrary, a cushion is formed both in the case of 14Hst5 and 48Hst5, and in the latter, an additional thick, diffuse, and highly hydrated layer of peptide and lipid molecules is formed, on top of the bilayer. Similar trends were observed from the simulations, which allowed us to hypothesize that positively charged patches of the amino acids lysine and arginine in all three peptides are essential for them to interact with and translocate over the bilayer. We therefore hypothesize that electrostatic interactions are important for the interaction between the solid-supported lipid bilayers and the peptide depending on the linear charge density through the primary sequence and the positively charged patches in the sequence. The understanding of how, why, and when the cushion is formed opens up the possibility for this system to be used in the research and development of new drugs and pharmaceuticals.

Deeper Insight of the Conformational Ensemble of Intrinsically Disordered Proteins.

It is generally known that, unlike structured proteins, intrinsically disordered proteins, IDPs, exhibit various structures and conformers, the so-called conformational ensemble, CoE. This study aims to better understand the conformers that make up the IDP ensemble by decomposing the CoE into groups separated by their radius of gyration, Rg. A common approach to studying CoE for IDPs is to use low-resolution techniques, such as small-angle scattering, and combine those with computer simulations on different length scales. Herein, the well-studied antimicrobial saliva protein histatin 5 was utilized as a model peptide for an IDP; the average intensity curves were obtained from small-angle X-ray scattering; and compared with fully atomistic, explicit water, molecular dynamics simulations; then, the intensity curve was decomposed with respect to the different Rg values; and their secondary structure propensities were investigated. We foresee that this approach can provide important information on the CoE and the individual conformers within; in that case, it will serve as an additional tool for understanding the IDP structure-function relationship on a more detailed level.

Relevance of amphiphilicity and helicity on the antibacterial action of a histatin 5-derived peptide.

Peptide dhvar4, derived from the active domain of our salivary peptide histatin 5, bears a Phe residue in the middle of its hydrophilic face when folded into an α-helix. We then synthesized an analog with this Phe replaced by Lys and two analogs preserving Phe but bearing two and three α-aminoisobutyric acid (Aib) residues to stabilize the helical structure. The aim of this design was to verify which of the two features is more favorable to the biological activity. We performed a conformational study by means of circular dichroism and nuclear magnetic resonance, made antibacterial tests, and assessed the stability of the peptides in human serum. We observed that amphiphilicity is more important than helix stability, provided a peptide can adopt a helical conformation in a membrane-mimetic environment.

Impact of metal coordination and pH on the antimicrobial activity of histatin 5 and the products of its hydrolysis.

This work focuses on the relationship between the coordination chemistry and antimicrobial activity of Zn(II) and Cu(II) complexes of histatin 5 and the products of its hydrolysis: its N-terminal fragment (histatin 5-8) and C-terminal fragment (histatin 8). Cu(II) coordinates in an albumin-like binding mode and Zn(II) binds to up to 3 His imidazoles. The antimicrobial activity of histatins and their metal complexes (i) strongly depends on pH - they are more active at pH 5.4 than at 7.4; (ii) the complexes and ligands alone are more effective in eradicating Gram-positive bacteria than the Gram-negative ones, and (iii) Zn(II) coordination is able to change the structure of the N-terminal region of histatin 5 (histatin 5-8) and moderately increase all of the studied histatins' antimicrobial potency.

Histatin 5-Inspired Short-Chain Peptides Selectively Combating Pathogenic Fungi with Multifaceted Mechanisms.

Short-chain antifungal peptides (AFPs) inspired by histatin 5 have been designed to address the problem of antifungal drug resistance. These AFPs demonstrate remarkable antifungal activity, with a minimal inhibitory concentration as low as 2 µg mL-1. Notably, these AFPs display a strong preference for targeting fungi rather than bacteria and mammalian cells. This is achieved by binding the histidine-rich domains of the AFPs to the Ssa1/2 proteins in the fungal cell wall, as well as the reduced membrane-disrupting activity due to their low amphiphilicity. These peptides disrupt the nucleus and mitochondria once inside the cells, leading to reactive oxygen species production and cell damage. In a mouse model of vulvovaginal candidiasis, the AFPs demonstrate not only antifungal activity, but also promote the growth of beneficial Lactobacillus spp. This research provides valuable insights for the development of fungus-specific AFPs and offers a promising strategy for the treatment of fungal infectious diseases.

Improving Photocleavage Efficiency of Photocleavable Protein for Antimicrobial Peptide Histatin 1 Expression.

BACKGROUND: Antimicrobial peptides (AMPs) are promising alternative agents for antibiotics to overcome antibiotic resistance problems. But, it is difficult to produce large-scale antimicrobial research due to the toxicity towards expression hosts or degradation by peptidases in the host. Therefore, heterologous recombinant expression of antimicrobial peptides has always been a challenging issue. OBJECTIVES: To overcome toxicity to the expression host and low expression level, a new photocleavable protein fusion expression method for antimicrobial peptides is provided.3 Methods: Through directed evolution and high throughput screening, a photocleavable protein mutant R6-2-6-4 with a higher photocleavage efficiency was obtained. The DNA coding sequence of antimicrobial peptide Histatin 1 was fused within the sequence of R6-2-6-4 gene. The fusion gene was successfully expressed in Escherichia coli expression system. RESULTS: Antimicrobial peptide Histatin 1 could be successfully expressed and purified by fusing within PhoCl mutant R6-2-6-4. The antimicrobial activity was rarely affected, and the MIC value was 33 ug/mL, which was basically equivalent to 32 ug/mL of the chemically synthesized Histatin 1. After amplification in a 5 L fermenter, the expression of PhoCl mutant (R6-2-6-4)-Histatin1 improved up to 87.6 mg/L in fermenter, and Histatin1 obtained by photocleavage also could up to 11 mg/L. The prepared Histatin1 powder remained stable when stored at 4oC for up to 4 months without any degradation. In addition, the expression and photocleavage of ß -Defensin105 and Lysostaphin verified the certain universality of the PhoCl mutant fusion expression system. CONCLUSION: Antimicrobial peptides Histatin 1, ß -Defensin 105 and Lysostaphin were successfully expressed and purified by photocleavable protein mutant. This may provide a novel strategy to express and purify antimicrobial peptides in the Escherichia coli expression system.

Tetrahedral DNA nanostructure improves transport efficiency and anti-fungal effect of histatin 5 against Candida albicans.

OBJECTIVES: Anti-microbial peptides (AMPs) have been comprehensively investigated as a novel alternative to traditional antibiotics against microorganisms. Meanwhile, Tetrahedral DNA nanostructures (TDNs) have gained attention in the field of biomedicine for their premium biological effects and transportation efficiency as delivery vehicles. Hence, in this study, TDN/Histatin 5 (His-5) was synthesized and the transport efficiency and anti-fungal effect were measured to evaluate the promotion of His-5 modified by TDNs. MATERIALS AND METHODS: Tetrahedral DNA nanostructures/His-5 complex was prepared via electrostatic attraction and characterized by transmission electron microscopy (TEM), polyacrylamide gel electrophoresis (PAGE), dynamic light scattering (DLS) and electrophoretic light scattering (ELS). The anti-fungal effect of the TDN/His-5 complex was evaluated by determining the growth curve and colony-forming units of C. albicans. The morphological transformation of C. albicans was observed by light microscope and scanning electron microscope (SEM). Immunofluorescence was performed, and potassium efflux was detected to mechanistically demonstrate the efficacy of TDN/His-5. RESULTS: The results showed that Histatin 5 modified by TDNs had preferable stability in serum and was effectively transported into C. albicans, leading to the increased formation of intracellular reactive oxygen species, higher potassium efflux and enhanced anti-fungal effect against C. albicans. CONCLUSIONS: Our study showed that TDN/His-5 was synthesized successfully. And by the modification of TDNs, His-5 showed increased transport efficiency and improved anti-fungal effect.

Full structural ensembles of intrinsically disordered proteins from unbiased molecular dynamics simulations.

Molecular dynamics (MD) simulation is widely used to complement ensemble-averaged experiments of intrinsically disordered proteins (IDPs). However, MD often suffers from limitations of inaccuracy. Here, we show that enhancing the sampling using Hamiltonian replica-exchange MD (HREMD) led to unbiased and accurate ensembles, reproducing small-angle scattering and NMR chemical shift experiments, for three IDPs of varying sequence properties using two recently optimized force fields, indicating the general applicability of HREMD for IDPs. We further demonstrate that, unlike HREMD, standard MD can reproduce experimental NMR chemical shifts, but not small-angle scattering data, suggesting chemical shifts are insufficient for testing the validity of IDP ensembles. Surprisingly, we reveal that despite differences in their sequence, the inter-chain statistics of all three IDPs are similar for short contour lengths (< 10 residues). The results suggest that the major hurdle of generating an accurate unbiased ensemble for IDPs has now been largely overcome.

Determining Rg of IDPs from SAXS Data.

There is a great interest within the research community to understand the structure-function relationship for intrinsically disordered proteins (IDPs); however, the heterogeneous distribution of conformations that IDPs can adopt limits the applicability of conventional structural biology methods. Here, scattering techniques, such as small-angle X-ray scattering, can contribute. In this chapter, we will describe how to make a model-free determination of the radius of gyration by using two different approaches, the Guinier analysis and the pair distance distribution function. The ATSAS package (Franke et al., J Appl Crystallogr 50:1212-1225, 2017) has been used for the evaluation, and throughout the chapter, different examples will be given to illustrate the discussed phenomena, as well as the pros and cons of using the different approaches.

Human salivary histatin-1 (Hst1) promotes bone morphogenetic protein 2 (BMP2)-induced osteogenesis and angiogenesis.

Large-volume bone defects can result from congenital malformation, trauma, infection, inflammation and cancer. At present, it remains challenging to treat these bone defects with clinically available interventions. Allografts, xenografts and most synthetic materials have no intrinsic osteoinductivity, and so an alternative approach is to functionalize the biomaterial with osteoinductive agents, such as bone morphogenetic protein 2 (BMP2). Because it has been previously demonstrated that human salivary histatin-1 (Hst1) promotes endothelial cell adhesion, migration and angiogenesis, we examine here whether Hst1 can promote BMP2-induced bone regeneration. Rats were given subcutaneous implants of absorbable collagen sponge membranes seeded with 0, 50, 200 or 500 µg Hst1 per sample and 0 or 2 µg BMP2 per sample. At 18 days postsurgery, rats were sacrificed, and implanted regional tissue was removed for micro computed tomography (microCT) analyses of new bone (bone volume, trabecular number and trabecular separation). Four samples per group were decalcified and subjected to immunohistochemical staining to analyze osteogenic and angiogenic markers. We observed that Hst1 increased BMP2-induced new bone formation in a dose-dependent manner. Co-administration of 500 µg Hst1 and BMP2 resulted in the highest observed bone volume and trabecular number, the lowest trabecular separation and the highest expression of osteogenic markers and angiogenic markers. Our results suggest that coadministration of Hst1 may enhance BMP2-induced osteogenesis and angiogenesis, and thus may have potential for development into a treatment for large-volume bone defects.

Salivary Histatin 1 and 2 Are Targeted to Mitochondria and Endoplasmic Reticulum in Human Cells.

Human salivary histatin 1 (Hst1) and Hst2 exhibit a series of cell-activating properties (e.g., promoting adhesion, spreading, migration and metabolic activity of mammalian cells). In contrast, Hst5 shows an anti-fungal property but no cell-activating properties. Previous findings suggest that their uptake and association with subcellular targets may play a determinant role in their functions. In this study, we studied the uptake dynamics and subcellular targets of Hst1, Hst2 and Hst5 in epithelial cells (HO1N1 human buccal carcinoma epithelial cell line). Confocal laser scanning microscopy (CLSM) revealed that fluorescently labeled Hst1 (F-Hst1) was taken up into the intracellular space of epithelial cells. Then, 60 min post-incubation, the total fluorescence of cell-associated F-Hst1, as measured using flow cytometry, was significantly higher compared to those of F-Hst2 and F-Hst5. In contrast, virtually no association occurred using the negative control-scrambled F-Hst1 (F-Hstscr). CLSM images revealed that F-Hst1, 2 and 5 co-localized with mitotrackerTM-labeled mitochondria. In addition, F-Hst1 and F-Hst2 but neither F-Hst5 nor F-Hst1scr co-localized with the ER-trackerTM-labeled endoplasmic reticulum. No co-localization of Hst1, 2 and 5 with lysosomes or the Golgi apparatus was observed. Furthermore, Hst1 and Hst2 but not Hst5 or Hst1scr significantly promoted the metabolic activity of both human epithelial cell lines, HaCaT human keratinocytes and primary human gingival fibroblasts.

Peptide Self-Assembly Is Linked to Antibacterial, but Not Antifungal, Activity of Histatin 5 Derivatives.

The rise of multidrug-resistant pathogens has awakened interest in new drug candidates such as antimicrobial peptides and their derivatives. Recent work suggests that some antimicrobial peptides have the ability to self-assemble into ordered amyloid-like nanostructures which facilitate their antibacterial activity. Here, we evaluate a histatin-based antimicrobial peptide, and its self-assembling derivative, in the interplay between self-assembly, membrane interactions, and antibacterial and antifungal activities. We demonstrate substantial membrane targeting by both peptides, as well as mechanistic insights into this mode of action, which correlates to their antifungal activity and is not affected by their self-assembling state. The ability to self-assemble does, however, significantly affect peptide antibacterial activity against both Gram-negative and Gram-positive bacteria. These results are surprising and hint at important distinctions between antifungal and antibacterial peptide activities in prokaryotes and eukaryotic microbes.IMPORTANCE Antimicrobial peptides are important modulators of host defense against bacterial, fungal, and viral pathogens in humans and other multicellular organisms. Two converging paradigms point to a link between antimicrobial peptides that self-assemble into amyloid-like nanoassemblies and classical amyloidogenic peptides that often have potent broad-spectrum antimicrobial activity, suggesting that antimicrobial and amyloidogenic peptides may represent two sides of the same coin. Here, we asked if the ability of an antifungal peptide to self-assemble affects its antifungal or antibacterial activity. We found that modifications of classical antifungal peptide derivative allowed it to self-assemble and did not alter its antifungal activity, and yet self-assembly substantially increased the antibacterial activity of the peptide. These results support the idea that peptide self-assembly can enhance antibacterial activities and emphasize a distinction between the action of antifungal peptides and that of antibacterial peptides. Accordingly, we suggest that the possible generality of this distinction should be widely tested.

The Interactions between the Antimicrobial Peptide P-113 and Living Candida albicans Cells Shed Light on Mechanisms of Antifungal Activity and Resistance.

In the absence of proper immunity, such as in the case of acquired immune deficiency syndrome (AIDS) patients, Candida albicans, the most common human fungal pathogen, may cause mucosal and even life-threatening systemic infections. P-113 (AKRHHGYKRKFH), an antimicrobial peptide (AMP) derived from the human salivary protein histatin 5, shows good safety and efficacy profiles in gingivitis and human immunodeficiency virus (HIV) patients with oral candidiasis. However, little is known about how P-113 interacts with Candida albicans or its degradation by Candida-secreted proteases that contribute to the fungi's resistance. Here, we use solution nuclear magnetic resonance (NMR) methods to elucidate the molecular mechanism of interactions between P-113 and living Candida albicans cells. Furthermore, we found that proteolytic cleavage of the C-terminus prevents the entry of P-113 into cells and that increasing the hydrophobicity of the peptide can significantly increase its antifungal activity. These results could help in the design of novel antimicrobial peptides that have enhanced stability in vivo and that can have potential therapeutic applications.

Anionic food color tartrazine enhances antibacterial efficacy of histatin-derived peptide DHVAR4 by fine-tuning its membrane activity.

Here it is demonstrated how some anionic food additives commonly used in our diet, such as tartrazine (TZ), bind to DHVAR4, an antimicrobial peptide (AMP) derived from oral host defense peptides, resulting in significantly fostered toxic activity against both Gram-positive and Gram-negative bacteria, but not against mammalian cells. Biophysical studies on the DHVAR4-TZ interaction indicate that initially large, positively charged aggregates are formed, but in the presence of lipid bilayers, they rather associate with the membrane surface. In contrast to synergistic effects observed for mixed antibacterial compounds, this is a principally different mechanism, where TZ directly acts on the membrane-associated AMP promoting its biologically active helical conformation. Model vesicle studies show that compared to dye-free DHVAR4, peptide-TZ complexes are more prone to form H-bonds with the phosphate ester moiety of the bilayer head-group region resulting in more controlled bilayer fusion mechanism and concerted severe cell damage. AMPs are considered as promising compounds to combat formidable antibiotic-resistant bacterial infections; however, we know very little on their in vivo actions, especially on how they interact with other chemical agents. The current example illustrates how food dyes can modulate AMP activity, which is hoped to inspire improved therapies against microbial infections in the alimentary tract. Results also imply that the structure and function of natural AMPs could be manipulated by small compounds, which may also offer a new strategic concept for the future design of peptide-based antimicrobials.

Effects of histatin 5 modifications on antifungal activity and kinetics of proteolysis.

Histatin 5 (Hst-5) is an antimicrobial peptide with strong antifungal activity against Candida albicans, an opportunistic pathogen that is a common cause of oral thrush. The peptide is natively secreted by human salivary glands and shows promise as an alternative therapeutic against infections caused by C. albicans. However, Hst-5 can be cleaved and inactivated by a family of secreted aspartic proteases (Saps) produced by C. albicans. Single-residue substitutions can significantly affect the proteolytic resistance of Hst-5 to Saps and its antifungal activity; the K17R substitution increases resistance to proteolysis, while the K11R substitution enhances antifungal activity. In this work, we showed that the positive effects of these two single-residue modifications can be combined in a single peptide, K11R-K17R, with improved proteolytic resistance and antifungal activity. We also investigated the effect of additional single-residue substitutions, with a focus on the effect of addition or removal of negatively charged residues, and found Sap-dependent effects on degradation. Both single- and double-substitutions affected the kinetics of proteolytic degradation of the intact peptide and of the fragments formed during degradation. Our results demonstrate the importance of considering proteolytic stability and not just antimicrobial activity when designing peptides for potential therapeutic applications.

Whole-Genome Approach to Understanding the Mechanism of Action of a Histatin 5-Derived Peptide.

The incidence of opportunistic fungal infections that threaten immunocompromised patients, along with the limited arsenal of antifungal drugs, calls for renewed efforts to develop novel antifungal therapies. Antimicrobial peptides have garnered interest as potential therapeutics. Among naturally occurring peptides, histatin 5 is a well-characterized 24-amino-acid peptide with strong antifungal activity. Our lab has identified a smaller histatin derivative, KM29, with stronger activity against multiple Candida spp., prompting us to investigate its fungicidal mechanism. A genetic screen was developed to test the Saccharomyces cerevisiae genomewide deletion collection for mutants with increased or decreased peptide sensitivity. The goal was to identify genes that would reveal insights into the mechanism of action of KM29, to be assessed in Candida albicans Several biological processes yielded increased sensitivity, with endosomal transport and vacuolar function appearing at high frequencies. Among the pathways involved in increased resistance, mitochondrial function showed the highest normalized genome frequency; hence, we focused on characterizing this pathway. KM29 localizes to mitochondria, and the killing activity depends on a functional electron transport chain. In addition, KM29 triggered reactive oxygen species (ROS) production, which was responsible for some cell death but insufficient to account for the complete killing activity. In agreement with this finding, we found that KM29 induced mitochondrial fragmentation and a mild loss of mitochondrial membrane potential. Furthermore, respiratory mutants exhibited severely diminished KM29 uptake. We confirmed this behavior in a C. albicans respiratory mutant. Taking our findings together, this work delineates the mitochondrial functions associated with KM29 fungicidal activity and provides additional pathways for further characterization in Candida spp.

Specific metallo-protein interactions and antimicrobial activity in Histatin-5, an intrinsically disordered salivary peptide.

Histatin-5 (Hst-5) is an antimicrobial, salivary protein that is involved in the host defense system. Hst-5 has been proposed to bind functionally relevant zinc and copper but presents challenges in structural studies due to its disordered conformation in aqueous solution. Here, we used circular dichroism (CD) and UV resonance Raman (UVRR) spectroscopy to define metallo-Hst-5 interactions in aqueous solution. A zinc-containing Hst-5 sample exhibits shifted Raman bands, relative to bands observed in the absence of zinc. Based on comparison to model compounds and to a family of designed, zinc-binding beta hairpins, the alterations in the Hst-5 UVRR spectrum are attributed to zinc coordination by imidazole side chains. Zinc addition also shifted a tyrosine aromatic ring UVRR band through an electrostatic interaction. Copper addition did not have these effects. A sequence variant, H18A/H19A, was employed; this mutant has less potent antifungal activity, when compared to Hst-5. Zinc addition had only a small effect on the thermal stability of this mutant. Interestingly, both zinc and copper addition shifted histidine UVRR bands in a manner diagnostic for metal coordination. Results obtained with a K13E/R22G mutant were similar to those obtained with wildtype. These experiments show that H18 and H19 contribute to a zinc binding site. In the H18A/H19A mutant the specificity of the copper/zinc binding sites is lost. The experiments implicate specific zinc binding to be important in the antimicrobial activity of Hst-5.

Dynamical Oligomerisation of Histidine Rich Intrinsically Disordered ProteinS Is Regulated through Zinc-Histidine Interactions.

Intrinsically disordered proteins (IDPs) can form functional oligomers and in some cases, insoluble disease related aggregates. It is therefore vital to understand processes and mechanisms that control pathway distribution. Divalent cations including Zn2+ can initiate IDP oligomerisation through the interaction with histidine residues but the mechanisms of doing so are far from understood. Here we apply a multi-disciplinary approach using small angle X-ray scattering, nuclear magnetic resonance spectroscopy, calorimetry and computations to show that that saliva protein Histatin 5 forms highly dynamic oligomers in the presence of Zn2+. The process is critically dependent upon interaction between Zn2+ ions and distinct histidine rich binding motifs which allows for thermodynamic switching between states. We propose a molecular mechanism of oligomerisation, which may be generally applicable to other histidine rich IDPs. Finally, as Histatin 5 is an important saliva component, we suggest that Zn2+ induced oligomerisation may be crucial for maintaining saliva homeostasis.

Temperature Dependence of Intrinsically Disordered Proteins in Simulations: What are We Missing?

The temperature dependence of the conformational properties in simulations of the intrinsically disordered model protein histatin 5 has been investigated using different combinations of force fields, water models, and atomistic and coarse-grained methods. The results have been compared to experimental data obtained from NMR, SAXS, and CD experiments to assess the accuracy and validity of the simulations. The results showed that neither simulations completely agreed with the experimental data, nor did they agree with each other. It was however possible to conclude that the observed conformational changes upon variations in temperature were not at all driven by electrostatic interactions. The final conclusion was that none of the simulations that were investigated in this study was able to accurately capture the temperature induced conformational changes of our model IDP.

Antimicrobial coatings prepared from Dhvar-5-click-grafted chitosan powders.

Antimicrobial peptides (AMP) are powerful components of the innate immune system, as they display wide activity spectrum and low tendency to induce pathogen resistance. Hence, the development of AMP-based coatings is a very promising strategy to prevent biomaterials-associated infections. This work aims to investigate if Dhvar-5-chitosan conjugates, previously synthesized by us via azide-alkyne "click" reaction, can be applied as antimicrobial coatings. Ultrathin coatings were prepared by spin coater after dissolving Dhvar-5-chitosan conjugate powder in aqueous acetic acid. Peptide orientation and exposure from the surface was confirmed by ellipsometry and contact angle measurements. Bactericidal activity was evaluated against Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, the most prevalent pathogens in implant-associated infections. Results showed that Dhvar-5-chitosan coatings displayed bactericidal effect. Moreover, since Dhvar-5 has head-to-tail amphipathicity, it was clear that the bactericidal potency was dependent on which domain of the peptide (cationic or hydrophobic) was exposed. In this context, Dhvar-5 immobilized through its C-terminus (exposing its hydrophobic end) presented higher antimicrobial activity against Gram-positive bacteria and reduced adhesion of Gram-negative bacteria. This orientation-dependent antimicrobial activity was further corroborated by the anti-biofilm assay, as covalent immobilization of Dhvar-5 through its C-terminus provided anti-biofilm properties to the chitosan thin film. Immobilization of Dhvar-5 showed no cytotoxic effect against HFF-1 cells, as both metabolic activity and cell morphology were similar to control. In conclusion, Dhvar-5-chitosan coatings are promising antimicrobial surfaces without cytotoxic effects against human cells. STATEMENT OF SIGNIFICANCE: AMP-tethering onto ground biomaterial is still a poorly explored strategy in research. In this work, AMP-tethered ground chitosan is used to produce highly antibacterial ultrathin films. Powdered AMP-tethered chitosan appears as an alternative solution for antimicrobial devices production, as it is suitable for large scale production, being easier to handle for fabrication of different coatings and materials with antimicrobial properties and without inducing toxicity.