Comparative antimicrobial profiles of Histatin 5, Histatin 8, and their copper complexes

Comparative antimicrobial profiles of Histatin 5, Histatin 8, and their copper complexes | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative antimicrobial profiles of Histatin 5, Histatin 8, and their copper complexes Danuta Witkowska, Justyna Sokołowska, Joanna Słowik, Katarzyna Zamłyńska, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6420699/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Histatins are histidine-rich antimicrobial peptides present in human saliva, with histatin 5 (Hst5) demonstrating the most potent antifungal activity. Previous studies have linked the antifungal properties of histatins, particularly those against Candida species, to their ability to bind metal ions such as Cu(II) and Zn(II). While the antimicrobial activity of some histatins is well established, the role of metal ion coordination in their mechanism of action remains an area of ongoing investigation. This study focuses on histatin 8 (Hst8), a less-explored member of the histatin family, and compares its metal-binding and antimicrobial properties to those of Hst5. Using isothermal titration microcalorimetry (ITC), we examined the interactions of Hst8 with Cu(II), Zn(II), and Ni(II) ions and evaluated its antimicrobial activity against Escherichia coli , Staphylococcus aureus and two Candida albicans strains. Our findings revealed significant differences in copper and zinc binding between Hst5 and Hst8, with both peptides exhibiting distinct antifungal profiles. These results highlight the potential role of metal ion coordination in modulating the antimicrobial efficacy of histatins, providing further insight into their therapeutic potential. histatin 5 histatin 8 copper (II) ions Isothermal titration calorimetry (ITC) binding mechanism antimicrobial activity Figures Figure 1 Figure 2 Figure 3 Figure 4 Key points Histatin 5 binds two Cu(II) ions; Histatin 8 binds only one with lower affinity. Cu(II) complexation enhances antifungal activity of Histatin 8 against Candida . Histatin peptides show pathogen-specific metal-dependent antimicrobial activity. Introduction The human microbiome is an intricate and dynamic system, in which even the slightest disruption in homeostasis can trigger a cascade of undesirable processes. Microorganisms interact not only with each other but also with human proteins, various biomolecules, and essential metal ions—all of which must be considered in these analyses. Microorganisms naturally residing in the human body often become the greatest threat to a weakened organism. With the growing resistance to antibiotics, there is an increasing focus on exploring human body components that, through enhancement, could serve as effective allies in this unequal battle. Human saliva and the gastrointestinal tract contain numerous compounds with antibacterial properties. While some have been extensively studied, others are still being discovered. Saliva comprises diverse groups of antimicrobial peptides and proteins, including mucins, proline-rich proteins (PRPs), cystatins, statherins, cathelicidins, and low-molecular-weight histatins. Bioactive peptides and proteins present in the human body have garnered significant interest from researchers seeking new antimicrobial drugs, as they exhibit low cytotoxicity and minimal side effects(Zolin et al., 2021 ). Some well-characterized antimicrobial peptides (AMPs) serve as strong foundations for structural modifications aimed at enhancing their effectiveness or stability. However, it is worth noting that modifying antimicrobial peptides to increase their activity against a specific pathogen may result in unpredictable and undesirable side effects(Groot et al., 2006 ). In vitro studies have confirmed the antifungal activity of histatins against various pathogenic fungi, particularly those from the genera Candida , Cryptococcus , and Aspergillus (Tsai & Bobek, 1997 ). Moreover, in HIV-infected patients with opportunistic Candida infections in the oral cavity, reduced salivary histatin levels have been observed, suggesting that their primary function may be to prevent oral candidiasis (Lin et al., 1999 ). Histatins are found in human saliva at concentrations ranging from 50 to 450 µM (Zolin et al., 2021 ) Many known histatins feature an NH₂-XXH motif, recognized as the ATCUN motif (Amino-Terminal Cu(II) and Ni(II) binding site), which binds copper(II) and nickel(II). In addition, they contain the Zn(II)-binding HEXXH motif, as well as polyhistidine-rich regions. The name of these compounds reflects their high histidine content in the primary structure. Studies have demonstrated that certain metal ions, particularly copper(II), can enhance the antimicrobial activity of peptides, including histatin 5 (Hst5) (Campbell et al., 2023 ). Cu(II) binding may increase Hst5 stability by protecting it from degradation by fungal proteases (Conklin et al., 2017 ). Some studies indicate that Zn(II) complexes with Hst5 exhibit greater antifungal properties than the peptide alone (Norris et al., 2020 ), whereas others suggest that Zn(II) can reduce Hst5 activity. It has been proposed that zinc exerts a concentration-dependent effect on biological activity of Hst5 (Campbell et al., 2022 ). Among the histatin family members, histatin 5 has the most potent antifungal activity identified to date, establishing it as a key molecule of interest in antimicrobial peptide research. Hst5 is a 24-amino-acid proteolytic fragment derived from histatin 3. Its amino acid sequence is presented in Fig. 1 . Produced and secreted by the sublingual, parotid, and submandibular glands, Hst5 is a natural and essential component of human saliva, where it plays a critical role in oral host defense mechanisms. Human saliva has also been shown to contain various metal ions, including zinc, copper, iron, nickel, and manganese, with Zn(II) being the most abundant (Norris et al., 2018 ). Our previous research has shown that histidine-rich peptides can bind multiple metal ions, including Cu(II), Ni(II), and Zn(II), through the imidazole side chains of histidine residues and other peptide functional groups (Migliorini et al., 2010 ; Witkowska et al., 2011 , 2012 ). Given this, and despite earlier reports suggesting that Hst5 binds copper at a 1:1 ratio, (Zolin et al., 2021 ) we sought to verify this hypothesis. To achieve this goal, we conducted studies at near-physiological pH using the isothermal titration microcalorimetry (ITC) technique. Despite extensive research, the antimicrobial mechanism of histatins remains incompletely understood. In particular, histatin 8 (Hst8) has been poorly characterized with respect to both its antimicrobial properties and its interactions with metal ions. It is reasonable to hypothesize that, like Hst5, Hst8 may also interact with copper ions through its NH₂-XXH motif or histidine residues. The presence of histidine-rich sequences in histatins suggests broader potential for metal binding, which could influence their biological activity and stability. Since Hst8 has not been extensively studied, several key research questions remain open. One of the primary uncertainties is how its interaction with metal ions differs from that of Hst5 in a physiological environment? Additionally, it is unclear whether these interactions influence the antimicrobial activity of Hst8 and whether Hst8 has antifungal or antibacterial properties comparable to those of Hst5. In this study, we investigated the interactions of Hst8 with copper, zinc, and nickel divalent ions, as well as its antimicrobial activity against two bacterial and two Candida strains, in direct comparison to Hst5. To ensure a robust and reliable evaluation, all experiments were conducted under identical conditions for both peptides, enabling a comprehensive assessment of their metal coordination properties and antimicrobial potential. We observed unexpected differences in the copper and zinc binding and antimicrobial properties of these histatins. In contrast, nickel ion binding showed only minor variations in coordination to these two peptides. The sequences of both tested peptides are presented in Fig. 1 . Materials and methods Isothermal titration calorimetry (ITC) ITC measurements were conducted at 25°C via a MicroCal PEAQ isothermal titration calorimeter. All reagents were obtained from Sigma-Aldrich and were of > 99% purity. The peptides (ordered from KareBay Biochem) were dissolved directly in a 20 mM MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid) buffer, pH 7.0. Metal ion stock solutions were prepared in deionized water (maximum conductivity of 0.06 µS/cm) at low pH (~ 2) in glass bottles. After the instrument was stabilized at 25°C, titrations were performed using 40 µL of metal ion solutions (approximately 2 mM for Cu(II) and 4 mM for Ni(II) and Zn(II) ions) to titrate 200 µL of histatin solutions, with an initial peptide concentration ten times lower than that of the metal ions. The titration consisted of 19 successive injections, with intervals of 150–180 seconds between each injection. Each assay was repeated a few times to ensure reproducibility. A background titration was subtracted from the results to account for the heat of dilution. The stirring rate was set at 750 rpm throughout the experiments, and the reference cell was filled with demineralized water. Data were processed using MicroCal PEAQ-ITC Analysis Software. An initial 0.4 µL injection was discarded from each data set to eliminate the effect of titrant diffusion across the syringe tip during the equilibration process. UV-Vis spectroscopy UV-Vis absorption spectra were recorded via a Cintra 3030 spectrophotometer (GBC Company) over the wavelength range of 200–800 nm. Quartz cuvettes with a 1 cm optical path length were used for all measurements. Peptide and Cu(II) solutions were prepared in MOPS buffer (20 mM, pH 7.0). The peptide concentration was maintained at 1 mM. The spectra were collected at 25°C following the incremental addition of Cu(II) ions to the peptide solution. The titration points included 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6 equivalents of Cu(II) relative to the peptide concentration. Each spectrum was recorded after allowing the solution to equilibrate for 5 minutes to ensure complex formation. Antimicrobial activity assay Determination of the minimum inhibitory concentration (MIC) The antimicrobial activity of histatins 5 and 8 and their complexes with Cu(II) was determined via a microdilution method in 96-well plate (Biologix) against the bacterial strains Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923, and the two yeast strains Candida albicans ATCC 90028 and C. albicans ATCC 10231 derived from the American Type Culture Collection according to the Clinical and Laboratory Standards Institute guidelines(Clsi, n.d.). The experiments were carried out in liquid medium Muller Hinton Broth (MHB) or Sabouraud Dextrose Broth (SDB)(BioMaxima S.A., Poland) for bacteria and yeast, respectively. Histatins were dissolved in sterile saline (0.9% NaCl) to obtain a concentration of 1 mg/ml and then a series of twofold dilutions was made in broth appropriate for the tested strains at the concentration ranging from 500 to 31 µg/ml. Additionally, saline with Cu(II) ions in concentration of 1 µM was used as a control for complexes with Cu(II). Ampicillin and fluconazole were used as positive controls for E. coli (at concentrations ranging from 1 to 16 µg/ml) and C. albicans (at concentrations ranging from 0.5 to 8 µg/ml), respectively. A broth-free medium was used as a control for microbial growth. The suspension of tested strains was adjusted to the 0.5 McFarland standard and diluted in appropriate medium to obtain a final density of 5 x 10 6 CFU/ml. The microplates were incubated at 37 °C for 18 h, microbial growth was read spectrophotometrically via a microplate reader ASYS UVM 340 (Biogenet). All experiments were performed in triplicate. The minimum inhibitory concentration (MIC) values were estimated as the lowest concentration of compounds that completely inhibited the microbial growth. Growth curve assay The determination of the growth curves of E. coli ATCC 25922 and C. albicans ATCC 90028 in the presence of histatins and their complexes with Cu was performed by measuring the optical density of microbial cultures at 600 nm (OD600) against a control containing medium using an Implen OD600 DiluPhotometer. Cultures of the tested strains were carried out in a volume of 3 ml of MHB or SDB. Histatins at a concentration of 200 µg/ml (concentration lower than the MIC value) were used, the controls were cultures of microorganisms without histatins and additionally cultures with Cu(II) ions at a concentration of 5 µl/ml (1 µMol). Microbial suspensions with a final density of 5 x 10 6 CFU/ml were used. Measurements were carried out for 8 h at 1-hour intervals, and then after 22 and 24 h. All experiments were performed in triplicate. Statistical analysis was performed using the Student's t -test (Statistica 13.3). Data are presented as mean ± standard deviation (Mean ± SD), with p < 0.05 considered statistically significant. Results ITC and UV-Vis spectroscopy Isothermal titration calorimetry (ITC) experiments were conducted to assess the strength of copper(II) interactions with Hst 5 and Hst 8, as well as to determine the binding stoichiometry, enthalpy, and entropy changes. Since this technique does not provide unconditional data, assays were conducted under identical conditions (pH, buffer, and temperature) to enable a reliable comparison. The results are presented in Table 1 and Fig. 2. Additionally, measurements with Ni(II) and Zn(II) were performed (see Supplementary Table S1 and Fig. S1 , and S2). Each assay was repeated multiple times, and the results presented in Table 1 and Table S1 represent the averages of the two best-fitting measurements. The conditional parameters were directly obtained from the ITC experiment by fitting binding isotherms using nonlinear least-squares analysis, which is based on a model that assumes a single set of identical binding sites (Makowska et al., 2015 ). However, for Cu(II)-Hst5, fitting to a single set of identical binding sites was not feasible, and a two-site binding model provided a significantly better fit. Table 1 Experimental (conditional) thermodynamic parameters for Cu(II) binding to Hst5 and Hst8, determined from ITC measurements in MOPSO buffer, 25 o C. Ligand \(\:{\varvec{K}}_{\varvec{D}\varvec{I}\varvec{T}\varvec{C}}\left[\varvec{\mu\:}\varvec{M}\right]\) ΔH ITC [kcal/mol] N ITC [sites] Hst5 0.18 \(\:\:\pm\:\:\) 0.05 − 5.60 \(\:\pm\:\:\) 0.35 0.64 \(\:\:\pm\:\:\) 0.01 14.75 \(\:\:\pm\:\) 1.24 − 13.95 \(\:\pm\:\:\) 0.32 0.83 \(\:\:\pm\:\:\) 0.03 Hst8 12.30 \(\:\:\pm\:\:\) 3.07 − 9.52 \(\:\pm\:\:\) 0.57 0.93 \(\:\:\pm\:\:\) 0.02 The results for the interaction of Cu(II) with histatin 5 revealed two binding sites: one with high affinity (0.18 µM) and another with moderate affinity (~ 14 µM). In contrast, the interaction between Cu(II) and histatin 8 resulted in a single set of binding sites with moderate affinity (12.3 µM). All interactions were enthalpically driven (Table 1 ). In the case of Zn(II) and Ni(II) interactions with Hst5 and Hst8, significantly weaker affinities were observed (ranging from 110 to 238 µM). These interactions were also enthalpically driven, with additional entropic contributions detected for Zn(II) binding to Hst8. Both histatin titrations with Ni(II) at the same pH revealed that only half of the expected binding sites were engaged in the interaction (n ≈ 0.5). In contrast, zinc interactions with the studied histatins exhibited distinct differences in stoichiometry and the underlying driving forces of the reaction (Table S1 , Fig. S2). UV-Vis spectroscopy was performed up to a Cu:Hst ratio of 1.6:1, with scans recorded after each addition of 0.2 equivalents of Cu(II). For the Cu-Hst5 complex, the maximum absorbance was observed at 520 nm for a 1:1 ratio, shifting slightly to 530 nm at a 1.6:1 ratio (Figure S3). These maxima indicate the formation of square-planar Cu-Hst5 complexes, where each Cu(II) ion is coordinated by four nitrogen atoms: one from the N-terminal amino group, two from amide groups, and one from the imidazole side chain of His3 (Conklin et al., 2017 ; Witkowska et al., 2011 ). During the formation of the Cu(II)-Hst8 complex, a maximum in the d-d transition region was observed at 515 nm for a Cu(II):Hst8 ratio of 1:1 (Figure S4, Table S2). This absorption is characteristic of Cu(II) coordination to four nitrogen atoms, similar to what was observed for Cu(II)-Hst5 (Conklin et al., 2017 ; Witkowska et al., 2011 ). Since the affinity of zinc (II) ions for Hst 5 and Hst 8 was weak (K D values of 238 and 195 µM, respectively), we focused on investigating whether Cu(II) ions enhance the antimicrobial activity of these histatins. MIC determination of histatins and their Cu(II) complexes The tested histatins and their copper complexes showed moderate antibacterial activity (Table 2 ). Hst5 showed anti-yeast activity at a concentration of 250 µg/ml, but did not inhibit bacterial growth (MIC > 500 µg/ml). Compared with the native preparations, the complexes of Hst5 with Cu(II) ions had greater antibacterial activity. The MIC value decreased twofold for E. coli ATCC 25922, reaching 250 µg/ml, and for S. aureus ATCC 25923, it reached 500 µg/ml. No greater activity of Hst5 copper complexes against the tested C. albicans strains was observed. Hst8 revealed the best antibacterial efficacy against E. coli ATCC 25922 at a concentration of 250 µg/ml, and was less active against the other microorganisms tested, for which the MIC values ​​were 500 µg/ml. In the case of the Hst8 copper complex, a decrease in the MIC value (from 500 to 250 µg/ml) was observed only for the C. albicans ATCC 10231 strain. Ampicillin used as antibacterial control inhibited the growth of E. coli ATCC 25922 and S. aureus ATCC 25923 at concentrations of 2 and 1 µg/ml, respectively. Fluconazole was effective against C. albicans ATCC 90028 (MIC of 4 µg/ml) and C. albicans ATCC 10231 (MIC of 8 µg/ml). Growth curve assay Kinetic growth inhibition analyses were carried out for E. coli ATCC 25922 and C. albicans ATCC 90028. Both tested histatins, Hst8 and Hst5, as well as their Cu(II) complexes at the concentration of 200 µg/ml, inhibited the growth of E. coli . Changes were noticeable as early as the 3rd hour of culture in the case of Hst5 and its complex (inhibition of 23 and 40%). This effect was observed in the following hours of incubation, and the 24 h growth inhibition rates were 29 and 24%, respectively (Fig. 3 ). Hst8 and its cooper complex showed antibacterial activity after 6 h of E. coli growth (inhibitions of 17 and 14%); the highest percentage of growth inhibition (30–31%) was observed after 22 h of incubation. Inhibition of the growth of C. albicans by histatins was observed after 4–8 hours of incubation. Hst5 and the Cu(II) complex were the most active at longer incubation times. After 22 and 24 hours of incubation, yeast growth inhibition of approximately 70% was observed for both preparations. The differences in yeast growth were not so clear after 22–24 hours for Hst8 and the Cu(II) complex. Hst8 was found to inhibit the growth of C. albicans to a small but statistically significant extent after 5 h of culturing, after which antimicrobial activity was not detected (p < 0.05). The copper complex Hst8 showed an inhibitory effect on the growth of C. albicans after 6 h (Fig. 4 ) compared to the culture containing Hst8 and the control (p 0.05) were found between the culture of E. coli in Mueller-Hinton medium (control) and the culture of the strain with the addition of copper ions (control + Cu(II)). Table 2 Antimicrobial activity of histatins and complexes with Cu(II) ions. MIC µg/ml E. coli 25922 S. aureus 25923 C. albicans 90028 C. albicans 10231 Hst5 > 500 > 500 250 250 Hst5 + Cu(II) 250 500 250 250 Hst8 250 500 500 500 Hst8 + Cu(II) 250 500 500 250 Ampicillin 2 1 - - Fluconazole - - 4 8 Discussion Histatin 5, like other polyhistidyl peptides, has been extensively studied. However, research on histatin 8 remains limited. A recent 2024 study investigated its interactions with metal ions and the antimicrobial properties of its complexes, using potentiometry and spectroscopic techniques to analyze histatin binding with copper and zinc ions at a maximum metal-to-peptide ratio of 1:1 (Dzień et al., 2024 ). In our study, we employed isothermal titration microcalorimetry (ITC) to study their interactions with Cu(II), Zn(II) and Ni(II) ions, which provides simultaneous insights into the stoichiometry, binding affinity, and thermodynamic driving forces of these interactions. Unlike potentiometric methods, ITC measurements are conducted in aqueous buffer under constant pH conditions (pH 7.0 here), closely mimicking the physiological environment of human saliva. This makes ITC a powerful tool for investigating interactions between natural products or their analogues (e.g., histatins) and metal ions, offering deeper insights into their fundamental binding mechanisms. As a complementary technique, we employed UV-Vis spectroscopy to elucidate the geometry of the Cu(II) and Ni(II) complexes with both histatins. There are discrepancies in the reported results of Cu(II) binding to Hst5. Some studies suggest that Hst5 has only a single binding site for copper ions (Dzień et al., 2024 ), whereas others indicate the presence of two distinct binding sites (Conklin et al., 2017 ). Brewer and Gilles, using electrospray ionization mass spectrometry (ESI-MS), demonstrated the presence of multiple binding sites for Cu(II) and Ni(II) ions in Hst5, whereas only a single binding site was identified for Zn(II) (Brewer & Lajoie, 2000 ). On the other hand, Gusman et al. suggested that histatin 5 possesses one binding site with high selectivity for zinc, and two sites with low affinity for this metal ions (Gusman et al., n.d.). Similarly, they demonstrated the presence of three binding sites on Hst5 for Cu(II) ions. Norris et al. demonstrated that the higher relative affinity of Hst5 for Zn(II) likely facilitates dimerization. All studies agree that zinc ions bind to Hst5 with lower affinity compared to nickel and copper ions. NMR studies have shown that the preferred binding site for Zn(II) is - 3 H-E-X-X-H 7 - (Dzień et al., 2024 ; Grogan et al., 2001 ). Cragnel and coworkers, using multiple techniques, confirmed that Zn(II) binding to Hst5 induces oligomer formation as the zinc ion concentration increases. Dimers are formed through imidazole-mediated binding of two Hst5 chains, maintaining a 1:1 (2:2) stoichiometry, while allowing for multiple distinct coordination modes (Cragnell et al., 2019 ). Most studies on Cu-Hst5 complexes indicate that copper binding enhances histatin antimicrobial activity, whereas only a few suggest a similar effect for zinc. It has been proposed that the formation of a Cu(II)-histatin complex is a prerequisite for the oxidative activity of Hst5 (Melino et al., 2014 ). The ability of histatins to bind metal ions—not only zinc and copper—may be a key property with diverse applications beyond their antimicrobial activity, such as sequestering and detoxifying toxic metals such as Ni(II). Bal and colleagues utilized potentiometry and spectrophotometry to investigate Ni(II) binding to Hst5, demonstrating strong coordination of one Ni(II) ion at its N-terminal site and weaker binding at the C-terminal site (Kurowska et al., 2011 )The binding geometry was square-planar, similar to that observed by others for Cu(II) ion coordination at the N-terminal region of Hst5. In our studies with histatin 5 and its shorter analogue –histatin 8, we identified two binding sites on Hst5 exclusively for copper ions. Nickel and zinc ions bind to Hst5 and Hst8 with approximately ten times weaker affinity. However, due to metal-buffer interactions, we can accurately compare only the differences in Hst5 and Hst8 interactions with each metal separately. Interestingly, copper ions bind to Hst5 in a distinctly different manner than to Hst8. Hst5 exhibits two sets of binding sites with dissociation constants (K DITC ) of 0.18 µM and 14.75 µM, whereas Hst8 has only one set of binding sites with a K DITC of 12.30 µM (Table 1 ). This difference is consistent with the sequence of Hst5, which, in addition to the ATCUN motif, contains two dihistidyl motifs and two additional histidine residues-potential anchoring sites. Hst8 has a single dihistidyl site, but its proximity to the ATCUN motif likely prevents the binding of a second copper atom or renders that interaction too weak to be detected under these measurement conditions. The proposed coordination modes of the Cu(II) complexes with the ATCUN motifs of both peptides are shown in Fig. S5. At pH 7.00, the differences in nickel binding between Hst5 and Hst8 are minimal, with Hst5 showing a slightly greater affinity for nickel ions (Table S1 ). Our studies revealed that these two peptides bind zinc in distinct ways. Although both complexes are relatively weak, the interaction of Hst5 with zinc is enthalpically driven, whereas in the case of Hst8, the entropic factor predominates (Table S1 ). Additionally, the stoichiometry of the binding is different for each peptide. The observed stoichiometry (N) of approximately 0.5 (Table S1 ) may be due to incomplete deprotonation of histidine residues at pH 7.0 or metal-mediated oligomerization, as previously demonstrated by Cragnel et al. for Zn(II)-Hst5 interactions (Figure S6). Biological activity The mechanism of action of antimicrobial peptides assumes their interaction with the pathogens' membrane. AMPs can bind to microorganisms directly through electrostatic interactions with anionic groups on bacterial cell membranes and then penetrate the bacterial cell membrane, resulting in their death. Some cell-penetrating peptides exhibit strong antibacterial activity across the cell membrane without disturbing the intracellular interaction mechanisms (Zhang et al., 2021 ). The mechanisms of action of Hst5 on C. albicans fungal cells suggest the involvement of many intracellular targets: nonlytic leakage of ATP and K + ions, mitochondrial damage and the generation of oxidative stress. Hst5 has been identified as the most potent histatin in inhibiting the growth of both yeast and filamentous forms of Candida (Du et al., 2017 ). Due to the nonspecific mechanisms of AMP activity, microorganisms have not developed resistance mechanisms, unlike many currently used antibiotics. Therefore, these compounds are among the most promising drug candidates for the development of new antibiotics (Zhang et al., 2021 ). The results of our studies confirmed the activity of Hst5 against strains C. albicans , whereas Hst8 showed lower anti-yeast activity (MICs of 250 and 500 µg/ml, respectively). The Hst8 copper complex showed greater activity (twice lower MIC value) against the C. albicans strain ATCC 10231, compared to the native peptide. Both histatines and copper complexes at concentrations below the MIC (200 µg/ml) apparently inhibited C. albicans growth after 22–24 hours of incubation compared with the control, but Hst5 and its complex with Cu had greater activity. Although our tests did not show any inhibition of the growth of the bacterial strains used, an increase in the antibacterial activity of the Hst5-copper complex was observed. Hst8 had the best antibacterial efficacy against E. coli ATCC 25922, but it also inhibited the growth of S. aureus (MICs of 250 and 500, respectively). No increase in antibacterial activity was demonstrated after Hst8 was complexed with Cu (II) ions. Du et al. ( 2017 ) described that Hst5 possesses high bactericidal activity against some pathogens that cause nosocomial infections. The MIC values for Gram-negative bacteria ( Acinetobacter baumanii, Pseudomonas aeruginosa , and Enterobacter sp.) ranged from 38 to 90 µMol, which corresponds to a concentration of 115,3–273 µg/ml Hst5. The results obtained by(Matheson et al., 2013 ) demonstrated a narrow spectrum of Hst8 anti-yeast activity against C. albicans and C. tropicalis (MICs of 1 and 5 mg/ml, respectively). However, methicillin-resistant Staphylococcus aureus (MRSA) and E. coli were not sensitive to this peptide. Other authors reported that His8 was active against drug-resistant strains of A. baumannii (MIC 32 µg/ml)(Neshani et al., 2020 ). Conclusion Our study provides new insights into the metal-binding properties of histatin 5 and histatin 8, highlighting significant differences in their interactions with Cu(II) and Zn(II) ions. ITC measurements revealed that Hst5 possesses two distinct binding sites for copper ions, whereas Hst8 has only one, reflecting differences in their sequences and structural motifs. The observed variations in metal affinity and thermodynamic parameters suggest that these peptides interact with metal ions in unique ways, which may influence their biological functions. Furthermore, our findings confirm the antifungal activity of Hst5 against C. albicans , with Hst8 displaying lower efficacy. Notably, complexation with copper increased the antifungal activity of Hst8, reducing the MIC value twofold. While neither peptide significantly inhibited bacterial growth, Hst8 showed the greatest antibacterial effect against E. coli and S. aureus . However, complexation with Cu(II) did not enhance its antibacterial properties. These results suggest that metal coordination plays a crucial role in modulating the biological activity of histatins, particularly in antifungal applications. The ability of Hst5 and its copper complex to effectively inhibit C. albicans highlights their potential as candidates for novel antimicrobial therapies. Further modifications of these peptides, guided by AI-based design, could enhance their antimicrobial activity and improve their stability, making them even more promising for therapeutic applications. Of course, in such applications one should remember about the synergistic effects of various proteins and peptides, as well as about interactions between microorganisms inhabiting a given area of ​​the human body and other conditions, e.g. concomitant diseases. Declarations Ethical Approval – not applicable Funding - not applicable Availability of data and materials – The raw data supporting the findings of this study are available from the corresponding author upon reasonable request. Author Contribution D.W. and J.K. wrote the main manuscript text, J.S., K.Z. and J.Sł. conducted experiments, D.W., J.K. and P.L. prepared figures. All authors reviewed the manuscript. Data Availability Data is provided within the manuscript and supplementary information files. References Brewer D, Lajoie G (2000) Evaluation of the metal binding properties of the histidine-rich antimicrobial peptides histatin 3 and 5 by electrospray ionization mass spectrometry. In COMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom (Vol. 14) Campbell JX, Gao S, Anand KS, Franz KJ (2022) Zinc Binding Inhibits Cellular Uptake and Antifungal Activity of Histatin-5 in Candida albicans . ACS Infect Dis 8(9):1920–1934. https://doi.org/10.1021/acsinfecdis.2c00289 Campbell JX, Schulte NB, Lai B, Harris HH, Franz KJ (2023) Histatin-5 interacts with cellular copper to promote antifungal activity against Candida albicans . Metallomics 15(12). https://doi.org/10.1093/mtomcs/mfad070 Clsi (n.d.). M07-A9: Methods for Dilution Antimicrobial Susceptibility Tests for Bacteria That Grow Aerobically; Approved Standard—Ninth Edition . Conklin SE, Bridgman EC, Su Q, Riggs-Gelasco P, Haas KL, Franz KJ (2017) Specific Histidine Residues Confer Histatin Peptides with Copper-Dependent Activity against Candida albicans . Biochemistry 56(32):4244–4255. https://doi.org/10.1021/acs.biochem.7b00348 Cragnell C, Staby L, Lenton S, Kragelund BB, Skepö M (2019) Dynamical oligomerisation of histidine rich intrinsically disordered proteins is regulated through zinc-histidine interactions. 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Journal of Virology , 80 (18), 9236–9243. https://doi.org/10.1128/jvi.00796-06 Gusman H, Lendenmann U, Grogan J, Troxler RF, Oppenheim FG (n.d.). Is salivary histatin 5 a metallopeptide? Kurowska E, Bonna A, Goch G, Bal W (2011) Salivary histatin-5, a physiologically relevant ligand for Ni(II) ions. J Inorg Biochem 105(9):1220–1225. https://doi.org/10.1016/j.jinorgbio.2011.06.002 Lin AL, Shi Q, Johnson DA, Patterson TF, Rinaldi MG, Yeh C-K (1999) Murphy Division, South Texas Veterans Health Care System (Vol. 6, Issue 6) Makowska J, Wyrzykowski D, Hirniak P, Uber D, Chmurzyński L (2015) Investigations of copper(II) complexation by fragments of the FBP28 protein using isothermal titration (ITC) and differential scanning calorimetry (DSC). J Therm Anal Calorim 121(1):263–268. https://doi.org/10.1007/s10973-015-4660-7 Matheson SMG, Cheeptham N, Huttunen-Hennelly HEK (2013) Investigating the Effects of Hydrophobicity and Charge on the Therapeutic Ability of the Antimicrobial Histatin 8 Peptide for Potential Use in Oral Applications. Int J Biology 5(2). https://doi.org/10.5539/ijb.v5n2p85 Melino S, Santone C, Di Nardo P, Sarkar B (2014) Histatins: Salivaryx peptides with copper(II)- and zinc(II)-binding motifs Perspectives for biomedical applications. FEBS J (Vol 281:657–672. https://doi.org/10.1111/febs.12612 . Blackwell Publishing Ltd Migliorini C, Witkowska D, Valensin D, Kamysz W, Kozlowski H (2010) Competition between histamine-like and poly-imidazole coordination sites for Cu2 + and Zn2 + ions in zebra-fish peptide of prion-like protein. Dalton Trans 39(37):8663–8670. https://doi.org/10.1039/c0dt00137f Neshani A, Sedighian H, Mirhosseini SA, Ghazvini K, Zare H, Jahangiri A (2020) Antimicrobial peptides as a promising treatment option against Acinetobacter baumannii infections. Microbial Pathogenesis , 146 . https://doi.org/10.1016/j.micpath.2020.104238 Norris HL, Friedman J, Chen Z, Puri S, Wilding G, Edgerton M (2018) Salivary metals, age, and gender correlate with cultivable oral Candida carriage levels. J Oral Microbiol 10(1). https://doi.org/10.1080/20002297.2018.1447216 Norris HL, Kumar R, Ong CY, Xu D, Edgerton M (2020) Zinc binding by histatin 5 promotes fungicidal membrane disruption in C. albicans and C. Glabrata . J Fungi 6(3):1–16. https://doi.org/10.3390/jof6030124 Tsai H, Bobek LA (1997) Studies of the Mechanism of Human Salivary Histatin-5 Candidacidal Activity with Histatin-5 Variants and Azole-Sensitive and-Resistant Candida Species (Vol. 41, Issue 10) Witkowska D, Bielinska S, Kamysz W, Kozlowski H (2011) Cu2 + and Ni2 + interactions with N-terminal fragments of Hpn and Hpn-like proteins from Helicobacter pylori : Unusual impact of poly-Gln sequence on the complex stability. J Inorg Biochem 105(2):208–214. https://doi.org/10.1016/j.jinorgbio.2010.11.004 Witkowska D, Valensin D, Rowinska-Zyrek M, Karafova A, Kamysz W, Kozlowski H (2012) Coordination of Ni 2 + and Cu 2 + to metal ion binding domains of E. coli SlyD protein. J Inorg Biochem 107(1):73–81. https://doi.org/10.1016/j.jinorgbio.2011.11.012 Zhang R, Fan X, Jiang X, Zou M, Xiao H, Wu G (2021) Multiple Mechanisms of the Synthesized Antimicrobial Peptide TS against Gram-Negative Bacteria for High Efficacy Antibacterial Action In Vivo . Molecules 26(1). https://doi.org/10.3390/MOLECULES26010060 Zolin GVS, Fonseca FH, da, Zambom CR, Garrido SS (2021) Histatin 5 metallopeptides and their potential against Candida albicans pathogenicity and drug resistance. In Biomolecules (Vol. 11, Issue 8). MDPI AG. https://doi.org/10.3390/biom11081209 Additional Declarations No competing interests reported. Supplementary Files HistatinsSupplementary29III2025.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6420699","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":454846221,"identity":"77eb647d-465a-432c-8dfa-a5d06e8e0bab","order_by":0,"name":"Danuta Witkowska","email":"data:image/png;base64,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","orcid":"","institution":"University of Opole","correspondingAuthor":true,"prefix":"","firstName":"Danuta","middleName":"","lastName":"Witkowska","suffix":""},{"id":454846222,"identity":"d5330b39-8c4d-462c-9ca4-092f87e3eeb9","order_by":1,"name":"Justyna Sokołowska","email":"","orcid":"","institution":"University of Opole","correspondingAuthor":false,"prefix":"","firstName":"Justyna","middleName":"","lastName":"Sokołowska","suffix":""},{"id":454846223,"identity":"96874e12-569f-41db-b086-56ae4da27141","order_by":2,"name":"Joanna Słowik","email":"","orcid":"","institution":"University of Opole","correspondingAuthor":false,"prefix":"","firstName":"Joanna","middleName":"","lastName":"Słowik","suffix":""},{"id":454846224,"identity":"85a08a63-340a-4096-8aea-06c141b7cd5c","order_by":3,"name":"Katarzyna Zamłyńska","email":"","orcid":"","institution":"Maria Curie- Skłodowska University","correspondingAuthor":false,"prefix":"","firstName":"Katarzyna","middleName":"","lastName":"Zamłyńska","suffix":""},{"id":454846225,"identity":"c3563557-af91-416c-a2a7-9e6f7fceea23","order_by":4,"name":"Jolanta Kutkowska","email":"","orcid":"","institution":"Maria Curie- Skłodowska University","correspondingAuthor":false,"prefix":"","firstName":"Jolanta","middleName":"","lastName":"Kutkowska","suffix":""},{"id":454846226,"identity":"4d22ed1c-0624-481e-8094-a8e4f4ed37e2","order_by":5,"name":"Paweł Lenartowicz","email":"","orcid":"","institution":"University of Opole","correspondingAuthor":false,"prefix":"","firstName":"Paweł","middleName":"","lastName":"Lenartowicz","suffix":""}],"badges":[],"createdAt":"2025-04-10 13:38:34","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6420699/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6420699/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82635927,"identity":"78b6818f-bffc-4b48-b455-fb7fa000885a","added_by":"auto","created_at":"2025-05-13 14:33:22","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":22445,"visible":true,"origin":"","legend":"\u003cp\u003eSequences of the tested peptides with corresponding abbreviations (shown on the right) used in the publication. The ATCUN-like motifs are highlighted with frames, whereas the histidine residues, the most likely binding sites for the studied divalent metal ions at physiological pH, are marked in red.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6420699/v1/3094ddb67556b7b7faced51a.png"},{"id":82635928,"identity":"17eab8dc-d275-4fb1-93f4-5dd10b376ff2","added_by":"auto","created_at":"2025-05-13 14:33:22","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":55441,"visible":true,"origin":"","legend":"\u003cp\u003eITC binding isotherms for the titration of Cu(II) into (A) Histatin 5 and (B) Histatin 8 in 20 mM MOPS, pH 7.0, at 25 °C. The top panels show the differential power signals recorded for each injection, whereas the bottom panels present the integrated peak areas, representing the heat released during each injection.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6420699/v1/c60b69465c1e77db4054105f.png"},{"id":82635930,"identity":"b557b25f-df75-440e-95c1-69d4931f13a4","added_by":"auto","created_at":"2025-05-13 14:33:22","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8918,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 in the presence of Hst5 and Hst8 and copper complexes at the concentration of 200 µg/ml (C - control, Hst5 – histatin 5, Hst5 Cu – histatin 5 Cu complex, Hst8 – histatin 8, Hst8 Cu – histatin 8 Cu complex)\u003c/p\u003e","description":"","filename":"Onlinedrawingimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6420699/v1/4d1a2f690f29c9f8e9b57518.png"},{"id":82635931,"identity":"9a1927d8-b756-4ebc-b1f1-ada87c738234","added_by":"auto","created_at":"2025-05-13 14:33:22","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":8240,"visible":true,"origin":"","legend":"\u003cp\u003eGrowth of \u003cem\u003eC. albicans\u003c/em\u003e ATCC 90028 in the presence of histatins 5 and 8 and copper complexes at the concentration of 200 µg/ml (C - control, Hst5 – histatin 5, Hst5 Cu – histatin 5 and Cu complex, Hst8 – histatin 8, Hst8 Cu – histatin 8 and Cu complex)\u003c/p\u003e","description":"","filename":"Onlinedrawingimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6420699/v1/d6731b05e095b416b4e211fb.png"},{"id":88359511,"identity":"7d08ad34-e8b1-4c32-8801-9d6199dc6306","added_by":"auto","created_at":"2025-08-05 15:53:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":746122,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6420699/v1/673c65e3-bc80-47db-8ecb-36146813ae1c.pdf"},{"id":82637160,"identity":"dc00bb0e-ef5a-4ff2-87cb-7381048b0bc8","added_by":"auto","created_at":"2025-05-13 14:41:22","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1999888,"visible":true,"origin":"","legend":"","description":"","filename":"HistatinsSupplementary29III2025.docx","url":"https://assets-eu.researchsquare.com/files/rs-6420699/v1/4a5a4816662938f8e3d71ebd.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative antimicrobial profiles of Histatin 5, Histatin 8, and their copper complexes","fulltext":[{"header":"Key points","content":"\u003cul\u003e\n \u003cli\u003eHistatin 5 binds two Cu(II) ions; Histatin 8 binds only one with lower affinity.\u003c/li\u003e\n \u003cli\u003eCu(II) complexation enhances antifungal activity of Histatin 8 against \u003cem\u003eCandida\u003c/em\u003e.\u003c/li\u003e\n \u003cli\u003eHistatin peptides show pathogen-specific metal-dependent antimicrobial activity.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"Introduction","content":"\u003cp\u003eThe human microbiome is an intricate and dynamic system, in which even the slightest disruption in homeostasis can trigger a cascade of undesirable processes. Microorganisms interact not only with each other but also with human proteins, various biomolecules, and essential metal ions\u0026mdash;all of which must be considered in these analyses. Microorganisms naturally residing in the human body often become the greatest threat to a weakened organism. With the growing resistance to antibiotics, there is an increasing focus on exploring human body components that, through enhancement, could serve as effective allies in this unequal battle.\u003c/p\u003e \u003cp\u003eHuman saliva and the gastrointestinal tract contain numerous compounds with antibacterial properties. While some have been extensively studied, others are still being discovered. Saliva comprises diverse groups of antimicrobial peptides and proteins, including mucins, proline-rich proteins (PRPs), cystatins, statherins, cathelicidins, and low-molecular-weight histatins. Bioactive peptides and proteins present in the human body have garnered significant interest from researchers seeking new antimicrobial drugs, as they exhibit low cytotoxicity and minimal side effects(Zolin et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSome well-characterized antimicrobial peptides (AMPs) serve as strong foundations for structural modifications aimed at enhancing their effectiveness or stability. However, it is worth noting that modifying antimicrobial peptides to increase their activity against a specific pathogen may result in unpredictable and undesirable side effects(Groot et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2006\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn vitro studies have confirmed the antifungal activity of histatins against various pathogenic fungi, particularly those from the genera \u003cem\u003eCandida\u003c/em\u003e, \u003cem\u003eCryptococcus\u003c/em\u003e, and \u003cem\u003eAspergillus\u003c/em\u003e (Tsai \u0026amp; Bobek, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1997\u003c/span\u003e). Moreover, in HIV-infected patients with opportunistic \u003cem\u003eCandida\u003c/em\u003e infections in the oral cavity, reduced salivary histatin levels have been observed, suggesting that their primary function may be to prevent oral candidiasis (Lin et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). Histatins are found in human saliva at concentrations ranging from 50 to 450 \u0026micro;M (Zolin et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e)\u003c/p\u003e \u003cp\u003eMany known histatins feature an NH₂-XXH motif, recognized as the ATCUN motif (Amino-Terminal Cu(II) and Ni(II) binding site), which binds copper(II) and nickel(II). In addition, they contain the Zn(II)-binding HEXXH motif, as well as polyhistidine-rich regions. The name of these compounds reflects their high histidine content in the primary structure. Studies have demonstrated that certain metal ions, particularly copper(II), can enhance the antimicrobial activity of peptides, including histatin 5 (Hst5) (Campbell et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Cu(II) binding may increase Hst5 stability by protecting it from degradation by fungal proteases (Conklin et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Some studies indicate that Zn(II) complexes with Hst5 exhibit greater antifungal properties than the peptide alone (Norris et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), whereas others suggest that Zn(II) can reduce Hst5 activity. It has been proposed that zinc exerts a concentration-dependent effect on biological activity of Hst5 (Campbell et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2022\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the histatin family members, histatin 5 has the most potent antifungal activity identified to date, establishing it as a key molecule of interest in antimicrobial peptide research. Hst5 is a 24-amino-acid proteolytic fragment derived from histatin 3. Its amino acid sequence is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Produced and secreted by the sublingual, parotid, and submandibular glands, Hst5 is a natural and essential component of human saliva, where it plays a critical role in oral host defense mechanisms. Human saliva has also been shown to contain various metal ions, including zinc, copper, iron, nickel, and manganese, with Zn(II) being the most abundant (Norris et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2018\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eOur previous research has shown that histidine-rich peptides can bind multiple metal ions, including Cu(II), Ni(II), and Zn(II), through the imidazole side chains of histidine residues and other peptide functional groups (Migliorini et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Witkowska et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). Given this, and despite earlier reports suggesting that Hst5 binds copper at a 1:1 ratio, (Zolin et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) we sought to verify this hypothesis. To achieve this goal, we conducted studies at near-physiological pH using the isothermal titration microcalorimetry (ITC) technique.\u003c/p\u003e \u003cp\u003eDespite extensive research, the antimicrobial mechanism of histatins remains incompletely understood. In particular, histatin 8 (Hst8) has been poorly characterized with respect to both its antimicrobial properties and its interactions with metal ions. It is reasonable to hypothesize that, like Hst5, Hst8 may also interact with copper ions through its NH₂-XXH motif or histidine residues. The presence of histidine-rich sequences in histatins suggests broader potential for metal binding, which could influence their biological activity and stability. Since Hst8 has not been extensively studied, several key research questions remain open. One of the primary uncertainties is how its interaction with metal ions differs from that of Hst5 in a physiological environment? Additionally, it is unclear whether these interactions influence the antimicrobial activity of Hst8 and whether Hst8 has antifungal or antibacterial properties comparable to those of Hst5.\u003c/p\u003e \u003cp\u003eIn this study, we investigated the interactions of Hst8 with copper, zinc, and nickel divalent ions, as well as its antimicrobial activity against two bacterial and two \u003cem\u003eCandida\u003c/em\u003e strains, in direct comparison to Hst5. To ensure a robust and reliable evaluation, all experiments were conducted under identical conditions for both peptides, enabling a comprehensive assessment of their metal coordination properties and antimicrobial potential. We observed unexpected differences in the copper and zinc binding and antimicrobial properties of these histatins. In contrast, nickel ion binding showed only minor variations in coordination to these two peptides. The sequences of both tested peptides are presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e "},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIsothermal titration calorimetry (ITC)\u003c/h2\u003e \u003cp\u003eITC measurements were conducted at 25\u0026deg;C via a MicroCal PEAQ isothermal titration calorimeter. All reagents were obtained from Sigma-Aldrich and were of \u0026gt;\u0026thinsp;99% purity. The peptides (ordered from KareBay Biochem) were dissolved directly in a 20 mM MOPSO (2-hydroxy-3-morpholinopropanesulfonic acid) buffer, pH 7.0. Metal ion stock solutions were prepared in deionized water (maximum conductivity of 0.06 \u0026micro;S/cm) at low pH (~\u0026thinsp;2) in glass bottles.\u003c/p\u003e \u003cp\u003eAfter the instrument was stabilized at 25\u0026deg;C, titrations were performed using 40 \u0026micro;L of metal ion solutions (approximately 2 mM for Cu(II) and 4 mM for Ni(II) and Zn(II) ions) to titrate 200 \u0026micro;L of histatin solutions, with an initial peptide concentration ten times lower than that of the metal ions. The titration consisted of 19 successive injections, with intervals of 150\u0026ndash;180 seconds between each injection. Each assay was repeated a few times to ensure reproducibility.\u003c/p\u003e \u003cp\u003eA background titration was subtracted from the results to account for the heat of dilution. The stirring rate was set at 750 rpm throughout the experiments, and the reference cell was filled with demineralized water. Data were processed using MicroCal PEAQ-ITC Analysis Software. An initial 0.4 \u0026micro;L injection was discarded from each data set to eliminate the effect of titrant diffusion across the syringe tip during the equilibration process.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eUV-Vis spectroscopy\u003c/h3\u003e\n\u003cp\u003eUV-Vis absorption spectra were recorded via a Cintra 3030 spectrophotometer (GBC Company) over the wavelength range of 200\u0026ndash;800 nm. Quartz cuvettes with a 1 cm optical path length were used for all measurements. Peptide and Cu(II) solutions were prepared in MOPS buffer (20 mM, pH 7.0). The peptide concentration was maintained at 1 mM. The spectra were collected at 25\u0026deg;C following the incremental addition of Cu(II) ions to the peptide solution. The titration points included 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, and 1.6 equivalents of Cu(II) relative to the peptide concentration. Each spectrum was recorded after allowing the solution to equilibrate for 5 minutes to ensure complex formation.\u003c/p\u003e\n\u003ch3\u003eAntimicrobial activity assay\u003c/h3\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDetermination of the minimum inhibitory concentration (MIC)\u003c/h2\u003e \u003cp\u003eThe antimicrobial activity of histatins 5 and 8 and their complexes with Cu(II) was determined via a microdilution method in 96-well plate (Biologix) against the bacterial strains \u003cem\u003eEscherichia coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e ATCC 25923, and the two yeast strains \u003cem\u003eCandida albicans\u003c/em\u003e ATCC 90028 and \u003cem\u003eC. albicans\u003c/em\u003e ATCC 10231 derived from the American Type Culture Collection according to the Clinical and Laboratory Standards Institute guidelines(Clsi, n.d.). The experiments were carried out in liquid medium Muller Hinton Broth (MHB) or Sabouraud Dextrose Broth (SDB)(BioMaxima S.A., Poland) for bacteria and yeast, respectively.\u003c/p\u003e \u003cp\u003eHistatins were dissolved in sterile saline (0.9% NaCl) to obtain a concentration of 1 mg/ml and then a series of twofold dilutions was made in broth appropriate for the tested strains at the concentration ranging from 500 to 31 \u0026micro;g/ml. Additionally, saline with Cu(II) ions in concentration of 1 \u0026micro;M was used as a control for complexes with Cu(II). Ampicillin and fluconazole were used as positive controls for \u003cem\u003eE. coli\u003c/em\u003e (at concentrations ranging from 1 to 16 \u0026micro;g/ml) and \u003cem\u003eC. albicans\u003c/em\u003e (at concentrations ranging from 0.5 to 8 \u0026micro;g/ml), respectively. A broth-free medium was used as a control for microbial growth.\u003c/p\u003e \u003cp\u003eThe suspension of tested strains was adjusted to the 0.5 McFarland standard and diluted in appropriate medium to obtain a final density of 5 x 10\u003csup\u003e6\u003c/sup\u003e CFU/ml. The microplates were incubated at 37 \u0026deg;C for 18 h, microbial growth was read spectrophotometrically via a microplate reader ASYS UVM 340 (Biogenet). All experiments were performed in triplicate.\u003c/p\u003e \u003cp\u003eThe minimum inhibitory concentration (MIC) values were estimated as the lowest concentration of compounds that completely inhibited the microbial growth.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eGrowth curve assay\u003c/h3\u003e\n\u003cp\u003eThe determination of the growth curves of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eC. albicans\u003c/em\u003e ATCC 90028 in the presence of histatins and their complexes with Cu was performed by measuring the optical density of microbial cultures at 600 nm (OD600) against a control containing medium using an Implen OD600 DiluPhotometer. Cultures of the tested strains were carried out in a volume of 3 ml of MHB or SDB. Histatins at a concentration of 200 \u0026micro;g/ml (concentration lower than the MIC value) were used, the controls were cultures of microorganisms without histatins and additionally cultures with Cu(II) ions at a concentration of 5 \u0026micro;l/ml (1 \u0026micro;Mol). Microbial suspensions with a final density of 5 x 10\u003csup\u003e6\u003c/sup\u003e CFU/ml were used. Measurements were carried out for 8 h at 1-hour intervals, and then after 22 and 24 h.\u003c/p\u003e \u003cp\u003eAll experiments were performed in triplicate. Statistical analysis was performed using the Student's \u003cem\u003et\u003c/em\u003e-test (Statistica 13.3). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD), with \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 considered statistically significant.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eITC and UV-Vis spectroscopy\u003c/h2\u003e \u003cp\u003eIsothermal titration calorimetry (ITC) experiments were conducted to assess the strength of copper(II) interactions with Hst 5 and Hst 8, as well as to determine the binding stoichiometry, enthalpy, and entropy changes. Since this technique does not provide unconditional data, assays were conducted under identical conditions (pH, buffer, and temperature) to enable a reliable comparison. The results are presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;2. Additionally, measurements with Ni(II) and Zn(II) were performed (see Supplementary Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e and Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, and S2). Each assay was repeated multiple times, and the results presented in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e represent the averages of the two best-fitting measurements. The conditional parameters were directly obtained from the ITC experiment by fitting binding isotherms using nonlinear least-squares analysis, which is based on a model that assumes a single set of identical binding sites (Makowska et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). However, for Cu(II)-Hst5, fitting to a single set of identical binding sites was not feasible, and a two-site binding model provided a significantly better fit.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eExperimental (conditional) thermodynamic parameters for Cu(II) binding to Hst5 and Hst8, determined from ITC measurements in MOPSO buffer, 25 \u003csup\u003eo\u003c/sup\u003eC.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLigand\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{\\varvec{K}}_{\\varvec{D}\\varvec{I}\\varvec{T}\\varvec{C}}\\left[\\varvec{\\mu\\:}\\varvec{M}\\right]\\)\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eΔH\u003csub\u003eITC\u003c/sub\u003e [kcal/mol]\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eN\u003csub\u003eITC\u003c/sub\u003e [sites]\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cb\u003eHst5\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.18\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.05\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;5.60 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.35\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.64\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.01\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14.75\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\)\u003c/span\u003e\u003c/span\u003e 1.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;13.95 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.32\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.83\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.03\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHst8\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e12.30\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e3.07\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;9.52 \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.57\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.93\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\pm\\:\\:\\)\u003c/span\u003e\u003c/span\u003e0.02\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe results for the interaction of Cu(II) with histatin 5 revealed two binding sites: one with high affinity (0.18 \u0026micro;M) and another with moderate affinity (~\u0026thinsp;14 \u0026micro;M). In contrast, the interaction between Cu(II) and histatin 8 resulted in a single set of binding sites with moderate affinity (12.3 \u0026micro;M). All interactions were enthalpically driven (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn the case of Zn(II) and Ni(II) interactions with Hst5 and Hst8, significantly weaker affinities were observed (ranging from 110 to 238 \u0026micro;M). These interactions were also enthalpically driven, with additional entropic contributions detected for Zn(II) binding to Hst8. Both histatin titrations with Ni(II) at the same pH revealed that only half of the expected binding sites were engaged in the interaction (n\u0026thinsp;\u0026asymp;\u0026thinsp;0.5). In contrast, zinc interactions with the studied histatins exhibited distinct differences in stoichiometry and the underlying driving forces of the reaction (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Fig. S2).\u003c/p\u003e\u003cp\u003eUV-Vis spectroscopy was performed up to a Cu:Hst ratio of 1.6:1, with scans recorded after each addition of 0.2 equivalents of Cu(II). For the Cu-Hst5 complex, the maximum absorbance was observed at 520 nm for a 1:1 ratio, shifting slightly to 530 nm at a 1.6:1 ratio (Figure S3). These maxima indicate the formation of square-planar Cu-Hst5 complexes, where each Cu(II) ion is coordinated by four nitrogen atoms: one from the N-terminal amino group, two from amide groups, and one from the imidazole side chain of His3 (Conklin et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Witkowska et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e). During the formation of the Cu(II)-Hst8 complex, a maximum in the d-d transition region was observed at 515 nm for a Cu(II):Hst8 ratio of 1:1 (Figure S4, Table S2). This absorption is characteristic of Cu(II) coordination to four nitrogen atoms, similar to what was observed for Cu(II)-Hst5 (Conklin et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Witkowska et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSince the affinity of zinc (II) ions for Hst 5 and Hst 8 was weak (K\u003csub\u003eD\u003c/sub\u003e values of 238 and 195 \u0026micro;M, respectively), we focused on investigating whether Cu(II) ions enhance the antimicrobial activity of these histatins.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMIC determination of histatins and their Cu(II) complexes\u003c/h3\u003e\n\u003cp\u003eThe tested histatins and their copper complexes showed moderate antibacterial activity (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Hst5 showed anti-yeast activity at a concentration of 250 \u0026micro;g/ml, but did not inhibit bacterial growth (MIC\u0026thinsp;\u0026gt;\u0026thinsp;500 \u0026micro;g/ml). Compared with the native preparations, the complexes of Hst5 with Cu(II) ions had greater antibacterial activity. The MIC value decreased twofold for \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922, reaching 250 \u0026micro;g/ml, and for \u003cem\u003eS. aureus\u003c/em\u003e ATCC 25923, it reached 500 \u0026micro;g/ml. No greater activity of Hst5 copper complexes against the tested \u003cem\u003eC. albicans\u003c/em\u003e strains was observed.\u003c/p\u003e \u003cp\u003eHst8 revealed the best antibacterial efficacy against \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 at a concentration of 250 \u0026micro;g/ml, and was less active against the other microorganisms tested, for which the MIC values ​​were 500 \u0026micro;g/ml. In the case of the Hst8 copper complex, a decrease in the MIC value (from 500 to 250 \u0026micro;g/ml) was observed only for the \u003cem\u003eC. albicans\u003c/em\u003e ATCC 10231 strain. Ampicillin used as antibacterial control inhibited the growth of \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eS. aureus\u003c/em\u003e ATCC 25923 at concentrations of 2 and 1 \u0026micro;g/ml, respectively. Fluconazole was effective against \u003cem\u003eC. albicans\u003c/em\u003e ATCC 90028 (MIC of 4 \u0026micro;g/ml) and \u003cem\u003eC. albicans\u003c/em\u003e ATCC 10231 (MIC of 8 \u0026micro;g/ml).\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGrowth curve assay\u003c/h2\u003e \u003cp\u003eKinetic growth inhibition analyses were carried out for \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922 and \u003cem\u003eC. albicans\u003c/em\u003e ATCC 90028.\u003c/p\u003e \u003cp\u003eBoth tested histatins, Hst8 and Hst5, as well as their Cu(II) complexes at the concentration of 200 \u0026micro;g/ml, inhibited the growth of \u003cem\u003eE. coli\u003c/em\u003e. Changes were noticeable as early as the 3rd hour of culture in the case of Hst5 and its complex (inhibition of 23 and 40%). This effect was observed in the following hours of incubation, and the 24 h growth inhibition rates were 29 and 24%, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Hst8 and its cooper complex showed antibacterial activity after 6 h of \u003cem\u003eE. coli\u003c/em\u003e growth (inhibitions of 17 and 14%); the highest percentage of growth inhibition (30\u0026ndash;31%) was observed after 22 h of incubation.\u003c/p\u003e \u003cp\u003eInhibition of the growth of \u003cem\u003eC. albicans\u003c/em\u003e by histatins was observed after 4\u0026ndash;8 hours of incubation. Hst5 and the Cu(II) complex were the most active at longer incubation times. After 22 and 24 hours of incubation, yeast growth inhibition of approximately 70% was observed for both preparations. The differences in yeast growth were not so clear after 22\u0026ndash;24 hours for Hst8 and the Cu(II) complex. Hst8 was found to inhibit the growth of \u003cem\u003eC. albicans\u003c/em\u003e to a small but statistically significant extent after 5 h of culturing, after which antimicrobial activity was not detected (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). The copper complex Hst8 showed an inhibitory effect on the growth of \u003cem\u003eC. albicans\u003c/em\u003e after 6 h (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e4\u003c/span\u003e) compared to the culture containing Hst8 and the control (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eNo statistically significant differences (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05) were found between the culture of \u003cem\u003eE. coli\u003c/em\u003e in Mueller-Hinton medium (control) and the culture of the strain with the addition of copper ions (control\u0026thinsp;+\u0026thinsp;Cu(II)).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eAntimicrobial activity of histatins and complexes with Cu(II) ions.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c5\" namest=\"c2\"\u003e \u003cp\u003eMIC \u0026micro;g/ml\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cem\u003eE. coli\u003c/em\u003e 25922\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eS. aureus\u003c/em\u003e 25923\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cem\u003eC. albicans\u003c/em\u003e 90028\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cem\u003eC. albicans\u003c/em\u003e 10231\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHst5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026gt;\u0026thinsp;500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHst5\u0026thinsp;+\u0026thinsp;Cu(II)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHst8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHst8\u0026thinsp;+\u0026thinsp;Cu(II)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e500\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e250\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAmpicillin\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFluconazole\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e-\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eHistatin 5, like other polyhistidyl peptides, has been extensively studied. However, research on histatin 8 remains limited. A recent 2024 study investigated its interactions with metal ions and the antimicrobial properties of its complexes, using potentiometry and spectroscopic techniques to analyze histatin binding with copper and zinc ions at a maximum metal-to-peptide ratio of 1:1 (Dzień et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn our study, we employed isothermal titration microcalorimetry (ITC) to study their interactions with Cu(II), Zn(II) and Ni(II) ions, which provides simultaneous insights into the stoichiometry, binding affinity, and thermodynamic driving forces of these interactions. Unlike potentiometric methods, ITC measurements are conducted in aqueous buffer under constant pH conditions (pH 7.0 here), closely mimicking the physiological environment of human saliva. This makes ITC a powerful tool for investigating interactions between natural products or their analogues (e.g., histatins) and metal ions, offering deeper insights into their fundamental binding mechanisms. As a complementary technique, we employed UV-Vis spectroscopy to elucidate the geometry of the Cu(II) and Ni(II) complexes with both histatins.\u003c/p\u003e \u003cp\u003eThere are discrepancies in the reported results of Cu(II) binding to Hst5. Some studies suggest that Hst5 has only a single binding site for copper ions (Dzień et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e), whereas others indicate the presence of two distinct binding sites (Conklin et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Brewer and Gilles, using electrospray ionization mass spectrometry (ESI-MS), demonstrated the presence of multiple binding sites for Cu(II) and Ni(II) ions in Hst5, whereas only a single binding site was identified for Zn(II) (Brewer \u0026amp; Lajoie, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). On the other hand, Gusman et al. suggested that histatin 5 possesses one binding site with high selectivity for zinc, and two sites with low affinity for this metal ions (Gusman et al., n.d.). Similarly, they demonstrated the presence of three binding sites on Hst5 for Cu(II) ions. Norris et al. demonstrated that the higher relative affinity of Hst5 for Zn(II) likely facilitates dimerization. All studies agree that zinc ions bind to Hst5 with lower affinity compared to nickel and copper ions. NMR studies have shown that the preferred binding site for Zn(II) is -\u003csup\u003e3\u003c/sup\u003eH-E-X-X-H\u003csup\u003e7\u003c/sup\u003e- (Dzień et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Grogan et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2001\u003c/span\u003e). Cragnel and coworkers, using multiple techniques, confirmed that Zn(II) binding to Hst5 induces oligomer formation as the zinc ion concentration increases. Dimers are formed through imidazole-mediated binding of two Hst5 chains, maintaining a 1:1 (2:2) stoichiometry, while allowing for multiple distinct coordination modes (Cragnell et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Most studies on Cu-Hst5 complexes indicate that copper binding enhances histatin antimicrobial activity, whereas only a few suggest a similar effect for zinc. It has been proposed that the formation of a Cu(II)-histatin complex is a prerequisite for the oxidative activity of Hst5 (Melino et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The ability of histatins to bind metal ions\u0026mdash;not only zinc and copper\u0026mdash;may be a key property with diverse applications beyond their antimicrobial activity, such as sequestering and detoxifying toxic metals such as Ni(II). Bal and colleagues utilized potentiometry and spectrophotometry to investigate Ni(II) binding to Hst5, demonstrating strong coordination of one Ni(II) ion at its N-terminal site and weaker binding at the C-terminal site (Kurowska et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e)The binding geometry was square-planar, similar to that observed by others for Cu(II) ion coordination at the N-terminal region of Hst5.\u003c/p\u003e \u003cp\u003eIn our studies with histatin 5 and its shorter analogue \u0026ndash;histatin 8, we identified two binding sites on Hst5 exclusively for copper ions. Nickel and zinc ions bind to Hst5 and Hst8 with approximately ten times weaker affinity. However, due to metal-buffer interactions, we can accurately compare only the differences in Hst5 and Hst8 interactions with each metal separately.\u003c/p\u003e \u003cp\u003eInterestingly, copper ions bind to Hst5 in a distinctly different manner than to Hst8. Hst5 exhibits two sets of binding sites with dissociation constants (K\u003csub\u003eDITC\u003c/sub\u003e) of 0.18 \u0026micro;M and 14.75 \u0026micro;M, whereas Hst8 has only one set of binding sites with a K\u003csub\u003eDITC\u003c/sub\u003e of 12.30 \u0026micro;M (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This difference is consistent with the sequence of Hst5, which, in addition to the ATCUN motif, contains two dihistidyl motifs and two additional histidine residues-potential anchoring sites. Hst8 has a single dihistidyl site, but its proximity to the ATCUN motif likely prevents the binding of a second copper atom or renders that interaction too weak to be detected under these measurement conditions. The proposed coordination modes of the Cu(II) complexes with the ATCUN motifs of both peptides are shown in Fig. S5. At pH 7.00, the differences in nickel binding between Hst5 and Hst8 are minimal, with Hst5 showing a slightly greater affinity for nickel ions (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Our studies revealed that these two peptides bind zinc in distinct ways. Although both complexes are relatively weak, the interaction of Hst5 with zinc is enthalpically driven, whereas in the case of Hst8, the entropic factor predominates (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Additionally, the stoichiometry of the binding is different for each peptide. The observed stoichiometry (N) of approximately 0.5 (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e) may be due to incomplete deprotonation of histidine residues at pH 7.0 or metal-mediated oligomerization, as previously demonstrated by Cragnel et al. for Zn(II)-Hst5 interactions (Figure S6).\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eBiological activity\u003c/h2\u003e \u003cp\u003eThe mechanism of action of antimicrobial peptides assumes their interaction with the pathogens' membrane. AMPs can bind to microorganisms directly through electrostatic interactions with anionic groups on bacterial cell membranes and then penetrate the bacterial cell membrane, resulting in their death. Some cell-penetrating peptides exhibit strong antibacterial activity across the cell membrane without disturbing the intracellular interaction mechanisms (Zhang et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). The mechanisms of action of Hst5 on \u003cem\u003eC. albicans\u003c/em\u003e fungal cells suggest the involvement of many intracellular targets: nonlytic leakage of ATP and K\u0026thinsp;+\u0026thinsp;ions, mitochondrial damage and the generation of oxidative stress. Hst5 has been identified as the most potent histatin in inhibiting the growth of both yeast and filamentous forms of \u003cem\u003eCandida\u003c/em\u003e (Du et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDue to the nonspecific mechanisms of AMP activity, microorganisms have not developed resistance mechanisms, unlike many currently used antibiotics. Therefore, these compounds are among the most promising drug candidates for the development of new antibiotics (Zhang et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe results of our studies confirmed the activity of Hst5 against strains \u003cem\u003eC. albicans\u003c/em\u003e, whereas Hst8 showed lower anti-yeast activity (MICs of 250 and 500 \u0026micro;g/ml, respectively). The Hst8 copper complex showed greater activity (twice lower MIC value) against the \u003cem\u003eC. albicans\u003c/em\u003e strain ATCC 10231, compared to the native peptide. Both histatines and copper complexes at concentrations below the MIC (200 \u0026micro;g/ml) apparently inhibited \u003cem\u003eC. albicans\u003c/em\u003e growth after 22\u0026ndash;24 hours of incubation compared with the control, but Hst5 and its complex with Cu had greater activity.\u003c/p\u003e \u003cp\u003eAlthough our tests did not show any inhibition of the growth of the bacterial strains used, an increase in the antibacterial activity of the Hst5-copper complex was observed. Hst8 had the best antibacterial efficacy against \u003cem\u003eE. coli\u003c/em\u003e ATCC 25922, but it also inhibited the growth of \u003cem\u003eS. aureus\u003c/em\u003e (MICs of 250 and 500, respectively). No increase in antibacterial activity was demonstrated after Hst8 was complexed with Cu (II) ions.\u003c/p\u003e \u003cp\u003eDu et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) described that Hst5 possesses high bactericidal activity against some pathogens that cause nosocomial infections. The MIC values for Gram-negative bacteria (\u003cem\u003eAcinetobacter baumanii, Pseudomonas aeruginosa\u003c/em\u003e, and \u003cem\u003eEnterobacter\u003c/em\u003e sp.) ranged from 38 to 90 \u0026micro;Mol, which corresponds to a concentration of 115,3\u0026ndash;273 \u0026micro;g/ml Hst5.\u003c/p\u003e \u003cp\u003eThe results obtained by(Matheson et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) demonstrated a narrow spectrum of Hst8 anti-yeast activity against \u003cem\u003eC. albicans\u003c/em\u003e and \u003cem\u003eC. tropicalis\u003c/em\u003e (MICs of 1 and 5 mg/ml, respectively). However, methicillin-resistant \u003cem\u003eStaphylococcus aureus\u003c/em\u003e (MRSA) and \u003cem\u003eE. coli\u003c/em\u003e were not sensitive to this peptide. Other authors reported that His8 was active against drug-resistant strains of \u003cem\u003eA. baumannii\u003c/em\u003e (MIC 32 \u0026micro;g/ml)(Neshani et al., \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusion","content":"\u003cp\u003eOur study provides new insights into the metal-binding properties of histatin 5 and histatin 8, highlighting significant differences in their interactions with Cu(II) and Zn(II) ions. ITC measurements revealed that Hst5 possesses two distinct binding sites for copper ions, whereas Hst8 has only one, reflecting differences in their sequences and structural motifs. The observed variations in metal affinity and thermodynamic parameters suggest that these peptides interact with metal ions in unique ways, which may influence their biological functions.\u003c/p\u003e \u003cp\u003eFurthermore, our findings confirm the antifungal activity of Hst5 against \u003cem\u003eC. albicans\u003c/em\u003e, with Hst8 displaying lower efficacy. Notably, complexation with copper increased the antifungal activity of Hst8, reducing the MIC value twofold. While neither peptide significantly inhibited bacterial growth, Hst8 showed the greatest antibacterial effect against \u003cem\u003eE. coli\u003c/em\u003e and \u003cem\u003eS. aureus\u003c/em\u003e. However, complexation with Cu(II) did not enhance its antibacterial properties.\u003c/p\u003e \u003cp\u003eThese results suggest that metal coordination plays a crucial role in modulating the biological activity of histatins, particularly in antifungal applications. The ability of Hst5 and its copper complex to effectively inhibit \u003cem\u003eC. albicans\u003c/em\u003e highlights their potential as candidates for novel antimicrobial therapies. Further modifications of these peptides, guided by AI-based design, could enhance their antimicrobial activity and improve their stability, making them even more promising for therapeutic applications. Of course, in such applications one should remember about the synergistic effects of various proteins and peptides, as well as about interactions between microorganisms inhabiting a given area of ​​the human body and other conditions, e.g. concomitant diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthical Approval\u0026nbsp;\u0026ndash; not applicable\u003c/p\u003e\n\u003cp\u003eFunding\u0026nbsp;- not applicable\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials \u0026ndash; The raw data supporting the findings of this study are available from the corresponding author upon reasonable request.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eD.W. and J.K. wrote the main manuscript text, J.S., K.Z. and J.Sł. conducted experiments, D.W., J.K. and P.L. prepared figures. All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eData is provided within the manuscript and supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBrewer D, Lajoie G (2000) Evaluation of the metal binding properties of the histidine-rich antimicrobial peptides histatin 3 and 5 by electrospray ionization mass spectrometry. In \u003cem\u003eCOMMUNICATIONS IN MASS SPECTROMETRY Rapid Commun. Mass Spectrom\u003c/em\u003e (Vol. 14)\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCampbell JX, Gao S, Anand KS, Franz KJ (2022) Zinc Binding Inhibits Cellular Uptake and Antifungal Activity of Histatin-5 in \u003cem\u003eCandida albicans\u003c/em\u003e. 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MDPI AG. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3390/biom11081209\u003c/span\u003e\u003cspan address=\"10.3390/biom11081209\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"histatin 5, histatin 8, copper (II) ions, Isothermal titration calorimetry (ITC), binding mechanism, antimicrobial activity","lastPublishedDoi":"10.21203/rs.3.rs-6420699/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6420699/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHistatins are histidine-rich antimicrobial peptides present in human saliva, with histatin 5 (Hst5) demonstrating the most potent antifungal activity. Previous studies have linked the antifungal properties of histatins, particularly those against \u003cem\u003eCandida\u003c/em\u003especies, to their ability to bind metal ions such as Cu(II) and Zn(II). While the antimicrobial activity of some histatins is well established, the role of metal ion coordination in their mechanism of action remains an area of ongoing investigation. This study focuses on histatin 8 (Hst8), a less-explored member of the histatin family, and compares its metal-binding and antimicrobial properties to those of Hst5. Using isothermal titration microcalorimetry (ITC), we examined the interactions of Hst8 with Cu(II), Zn(II), and Ni(II) ions and evaluated its antimicrobial activity against \u003cem\u003eEscherichia coli\u003c/em\u003e, \u003cem\u003eStaphylococcus aureus\u003c/em\u003e and two \u003cem\u003eCandida\u003c/em\u003e \u003cem\u003ealbicans\u003c/em\u003e strains. Our findings revealed significant differences in copper and zinc binding between Hst5 and Hst8, with both peptides exhibiting distinct antifungal profiles. These results highlight the potential role of metal ion coordination in modulating the antimicrobial efficacy of histatins, providing further insight into their therapeutic potential.\u003c/p\u003e","manuscriptTitle":"Comparative antimicrobial profiles of Histatin 5, Histatin 8, and their copper complexes","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 14:33:17","doi":"10.21203/rs.3.rs-6420699/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"928bdcce-b07c-4ba4-8e2e-198c9be2b41c","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-05T15:53:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-13 14:33:17","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6420699","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6420699","identity":"rs-6420699","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}