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Rational design of short peptide variants by using KunitzinRE, an amphibian-derived bioactivity peptide, for acquired potent broad-spectrum antimicrobial and improved therapeutic potential of commensalism coinfection of pathogens Zhanyi Yang, Shiqi He, Jiajun Wang, Yi Yang, Licong Zhang, Yanbing Li, and Anshan Shan J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00149 • Publication Date (Web): 08 Apr 2019 Downloaded from http://pubs.acs.org on April 9, 2019

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Journal of Medicinal Chemistry

Rational design of short peptide variants by using KunitzinRE, an amphibian-derived bioactivity peptide, for acquired potent broad-spectrum antimicrobial and improved therapeutic potential of commensalism coinfection of pathogens Zhanyi Yang1, Shiqi He1, Jiajun Wang1, Yi Yang1, Licong Zhang1, Yanbing Li2, Anshan Shan1* 1.Institute of Animal Nutrition, Northeast Agricultural University, Harbin 150030, P. R. China 2.College of Animal Science and Veterinary Medicine, Bayi Agricultural University, Daqing 163000, P. R. China

Abstract Commensalism coinfection of pathogens have seriously jeopardized human health. Currently, Kunitzin-RE as an amphibian-derived bioactivity peptide, is regarded as potential antimicrobial candidate. However, its antimicrobial properties were unsatisfactory. In this study, a set of shortened variants of Kunitzin-RE was developed by interception of peptide fragment and single site-mutation to investigate the effect of chain

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length, positive charge, hydrophobicity, amphipathicity and secondary structure on antimicrobial properties. Among them, W8 (AARIILRWRFR) significantly broadened the antimicrobial spectrum and showed the highest antimicrobial activity (GMall = 2.48µM) against all fungi and bacteria tested. Additionally, W8 showed high cell selectivity and salt tolerance in vitro while showing high effective against mice keratitis cause by infection by C. albicans 2.2086. Additionally, it also had obviously LPS-binding ability and a potent membrane-disruptive mechanism. Overall, these findings contributed to the design of short AMPs and to combat with the serious threat of commensalism coinfection of pathogens.

Key words: Antimicrobial peptides; Microbial infections; Broad-spectrum; Keratitis model; Membrane-disruptive mechanism;


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1. Introduction

Surgical site infections (SSIs) have become the third most continual healthcare-associated infection (HAI), resulting in the potential risk of morbidity and vastly prolonging hospital length of stay and tremendously consuming health care resources1-3. Currently, clinical studies recently demonstrated that all SSIs were primarily formed by commensalism coinfections via multimicrobial communities, including Staphylococcus aureus4, 5, Pseudomonas aeruginosa6, Candida albicans and Escherichia coli. Moreover, presurgical incisional asepsis utilizing antibiotics or the synergistic effects of different antibiotics at the site of infection is the most frequent and efficient method. However, antibiotic, as the representative drug for the treatment of microbial infectious diseases, they display better antimicrobial activity and efficiency, but their bactericidal mechanism can easily induce bacterial resistance7, 8. Currently, serious antibiotics abuse and the development of an antimicrobial agent that lags behind the



evolution of the bacterium result in many multidrug-resistant bacterial species such as Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-Resistant Enterococcus (VRE) or penicillin resistant streptococcus pneumoniae (PRSP) being found in a patient’s surgical incisional infection9, 10. These findings demonstrated that with the drug resistance bacteria and fungi gradually grow, pathogens increasingly are difficult to clear, increasing morbidity and making infectious diseases become more complicated with a range of antibiotics that has almost lost curative effects. Therefore, it is extremely urgent to obtain new categories of antimicrobial agents that have the characteristics of broad-spectrum activity, high killing efficiency, and low hemolytic activity and drug resistance. Antimicrobial peptides (AMPs) have gained much attention as potential antimicrobial agent templates based on their inhibition capacity of drug-resistant bacteria and superior killing efficiency within 5-60 min. It is difficult to guide microorganisms to develop drug resistance via


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special physical sterilization mechanisms of disruption and breaking liposomal membranes, ultimately inducing the leakage of cell contents and necrocytosis11-13. Additionally, some literature sources have further reported that AMPs possess some other functions, including suppressing biofilm formation, mediating the immune system response and promoting wound healing14, 15. These characteristics fundamentally compensate the fatal disadvantages of antibiotics. The source of AMPs is extensive and the naturally occurring AMPs are the main way for application of AMPs due to their good biosafety and low cost for large scale manufacturing compared with de novo synthetic peptides. Currently, Amphibian-derived AMPs account for more than onethird of almost 3000 naturally occurring peptides, and 90% of amphibiansderived AMPs are derived from frogs and toads. Their bioactivity may have guarded frog survival during their long development over millions of years, excluding changes in morphology16-19. Meanwhile, most of amphibians-derived AMPs mainly are released onto the skin surface to



prevent wound infection by pathogens, and prevent malaria and provide potent vasodilator activity via the contraction of smooth muscle and improvement of blood circulation and the skin extracts have been employed as the traditional medicine for centuries, such as Chinese traditional medicine to nurse bodily function and Egyptian medicine to treat wound infection and diarrhea20. Therefore, the capacity of amphibianderived skin AMPs deserves to be tapped for developing new therapeutic agents. Kunitzin-RE, a type of amphibian dermal granular or poison gland secretion of complicated bioactivity protein, was demonstrated to provide appreciably strong protection against predators and pathogens. However, the utilization value of Kunitzin-RE has restrictions in clinical applications due to high hemolytic, host toxicities and relatively weak antimicrobial activity. Particularly, Kunitzin-RE has only narrow-spectrum activity against E. coli, but not S. aureus and fungi 21, 22. Thus, simplification and design of variants of natural Kunitzin-RE is the optimal way to obtain safe and efficient novel antimicrobial agents.


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In this study, we designed a set of short peptide variants by interception of peptide fragment, increasing positive charge and crucial site-mutation of Kunitzin-RE based on its structural characteristics and residue distribution. Aiming at the design of peptides. Firstly, the 11residue peptide OT, truncated with the C-terminal six-residue loop (CKAAFC) with an inactive sequence, was obtained to simplify the peptide sequences and reduce the synthesis costs. Secondly, P8 was further designed by respectively replacing the K, N, K of the 3, 7, and 9 positions of OT with R to improve cationic ability. Consequently, a series of short peptide variants was systematically designed by replacing the P residue at the 8 position of P8 with different types of amino acid residues, such as aromatic hydrophobic residue (W, F, Y), aliphatic hydrophobic residue (G, A, I, L), polar residues (R, D, T) and amide residue (N, P), respectively. Finally, we selected the excellent peptide to transform the amphipathicity without a change in the hydrophobic and positive charges. Meanwhile, the in vitro bioactivity and cell selectivity of short peptide variants were



evaluated to screen the optimal AMPs. Consequently, the in vivo experiment was performed to assess the value of therapy applications and its structure-activity relationships and antimicrobial mechanism were further studied via circular dichroism (CD) spectra and electron microscopy.

2 Results 2.1 Characterization of peptides The molecular masses and secondary structure of peptides were accurately confirmed by MALDI-TOF MS and HPLC experimental analysis (Figures S1 and S2). The characterizations of the peptide are shown in Table 1. The theoretical molecular mass values of the peptide were almost consistent with their measured molecular mass values, suggesting that all the engineered peptides were successfully synthesized. Furthermore, the different hydrophobicity (H) values of Kunitzin-RE and engineered peptides derived from Kunitzin-RE were demonstrated, revealing the following hydrophobicity order: W8> I8> F8> L8> Y8> P8>


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A8> T8> G8> N8> D8> R8. Additionally, the hydrophobicity of W8 was consistent with its perfectly amphipathic derivatives TSW-1 and TSW-2. The wheel diagram (Figure 1 and S3) and relative hydrophobic moments demonstrated that TSW-1 (µHrel=0.652) and TSW-2 (µHrel=0.662) had the best arrangement with a completely integrated hydrophobic face and cationic face. Other engineered peptides had an almost identical proportionate between the hydrophobic and hydrophilic phases and relative hydrophobic moments, which were between 0.206 and 0.275, excluding that OT (µHrel=0.175). 2.2 Hemolytic analysis and cytotoxicity Hemolysis and cytotoxicity were important measurement indexes that verified the drug safety for clinical application. The hemolytic reactions of all peptides are shown in Figure 2C. Among these peptides, melittin and Kunitzin-RE were expressed at approximately more than 94% and 20% of the hemolytic rates at a peptide concentration of 128 μM, respectively.



Moreover, it was satisfactory that the other peptides induced inappreciable hemolytic activity with MHCs of less than 5% at all concentrations. Furthermore, the cytotoxicity of the peptide against RAW 264.7 macrophage cells was assessed (Figure 2A). Except for melittin and TSW1, the other engineered peptides maintained an approximately 90-100% cell survival rate at all concentrations, suggesting that most peptides had negligible toxicity and better biocompatibility. Interestingly, although hemolysis of TSW-1 did not cause the leakage of erythrocytes, it exhibited quite stronger cell cytotoxicity and eliminated the cell living rate over 89.76% at the highest concentration. 2.3 Antimicrobial Activity The antimicrobial efficacy of engineered peptides or antibiotics was calculated and summarized by the minimum inhibitory concentrations (MICs), minimum bactericidal concentrations and minimum fungicidal concentrations (MBCs/MFCs), as shown in Table 2. First, Kunitzin-RE only exhibited weaker antimicrobial activity, with MICs ranging from 32


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to 64 μM against most Gram-negative bacteria and was ineffective at inhibiting the growth of Gram-positive strains and fungal strains. However, most of the derivative peptides, excluding OT, P8, D8, and N8, exhibited wide antimicrobial spectra against bacteria and fungus. Among these peptides, the perfect amphipathic peptides (TSW-1 and TSW-2) and P8 analogs with different hydrophobicities by single amino acid substitution of I, A, F, L and W, demonstrated higher antimicrobial potency across bacterial species. Furthermore, these representative antibiotics had excellent antibacterial effects, but they did not exhibit significant broadspectrum antimicrobial capacity against bacteria and fungus. Additionally, the geometric means (GMs) presented in Table 3 evaluated the average antimicrobial ability of the peptides. For the P8 series analogs, the I, A, F, L and W substituted variants exhibited higher broad-spectrum sterilization ability, and the GM values were lower by 19.04 to 33.47-fold than those for Kunitzin-RE, especially peptide W8. Additionally, the perfectly amphipathic TSW-1 (GM

all=

4.97 µM), TSW-2 (GM


all=

3.67 µM)


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transformed from W8, was increased approximately 1.48-2 times the GMs against all bacterial species comparable to W8. In brief, W8 displayed the greatest antimicrobial effect, especially for Gram-positive strains, and the MICs of W8 (GM gram-positive= 1.19 µM; GM all= 2.48 µM) were even better or close than that of melittin (GM gram-positive= 2.83 µM; GM all= 2.28 µM). Furthermore, the MBC/MFC of peptides usually were higher, approximately 2-4 times comparable to their MICs. The therapeutic index (TI), which is the ratio of MHC to GM of MICs, was used to determine the cell selectivity of drugs and comprehensive clinical value. Among these peptides, W8 had the highest TIs (TI

all=

103.10; TI gram-positive= 215.30), nearly 264.36 times and 717.67 times that of Kunitzin-RE (TI all= 0.39; TI gram-positive= 0.30), and its TIs also showed excellent performance comparable with melittin (TI all= 0.44; TI gram-positive= 0.35). Interestingly, the TIs of perfectly amphipathic TSW-1 (TI all= 51.54; TI gram-positive= 45.25) and TSW-2 (TI all= 69.79; TI gram-positive= 53.82) were significantly lower than those of W8.


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Next, W8, as the best candidate among these peptides, was further studied regarding its efficiency of killing bacteria, as measured by its killing kinetics and the melittin as positive control. W8, at a concentration of 1 × MBC/MFC values, continuously increased the exposure time of E. coli 25922, S. aureus 29213 and C. albicans cgmcc2.2086. The results from Figure 2B indicated that W8 served to eliminate the growth of 99.99% of bacterial cells within 30 min and fungi cells within 3 min against these tested strains, which was a higher sterilization efficiency, but the sterilization efficiency of W8 still was lower than melittin. Finally, the drug resistance test was conducted to evaluate whether bacteria easily developed resistance to the W8. Serial 30 passages of P. aeruginosa 27853 in sub-MBC concentrations of W8 generated strains that remained highly sensitive to the W8, despite the truth that P. aeruginosa strains can quickly acquire drug resistance through antibiotic stimulation (Figure 2D). The results indicated that W8 hardly generated the drug resistance. In contrast, the gentamicin treatment group weakened the



sensitivity of P. aeruginosa strains as early as the 17th generation and its MBCs increased by 32-fold after 30 passages, it has shown that gentamicin treatment group can induce the development of drug resistance. Interestingly, melittin has similar effects to gentamicin as a positive control, its MBCs increased by 128-fold after 30 passages. However, as shown in Table S2, it was gratifying that W8 still maintains high efficiency against gentamicin-resistant P. aeruginosa and melittin-resistant P. aeruginosa. 2.4 Salt Sensitivity Assays The salt tolerance of the engineered peptides is an important safeguard to maintain the antimicrobial activity of peptides. Thus, the salt sensitivity of the engineered peptides was tested at the physiological concentrations of different salts (Table 4, 5, 6). the MICs of all peptides were partially improved by the addition of cations at physiological concentrations. Particularly, Na+ and Ca2+ sharply weakened the antimicrobial activity of most peptides, with the MICs increasing up to 16 - 32 folds against bacteria and fungi, respectively. Fortunately, various salts (K+, Zn2+, Mg2+, NH4+


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and Fe3+) at physiological concentrations showed little or no effect on the antimicrobial effect of W8, while the antimicrobial activity of W8 was relatively weakened by the addition of Na+ and Ca2+, corresponding with MICs increasing approximately 4 times against S. aureus 29213 or C. albicans 2.2086, respectively. By contrast, compared with W8, monovalent (Na+) can induce the compromise of the salt ion stability of TSW-1, with the MICs increasing by 4- and 16- fold against fungi and bacteria. Thus, these results revealed that W8 has strong salt tolerance compared with the other engineered peptides. Additionally, W8, with an imperfectly amphipathic structure, preferably resisted the salt disturbance at physiological concentrations compared with perfectly amphipathic peptides (TSW-1 or TSW-2). 2.5 In vivo antibacterial peptide potency against the keratitis model To determine whether the peptides still maintained their antibacterial potency in vivo, W8, with the highest therapeutic index (TI), was chosen to measure its antimicrobial efficiency in a mouse keratitis model infected



by C. albicans 2.2086. The amphotericin B was used as a positive control based on rapid sterilization efficiency against C. albicans 2.2086. MICs and MBCs of W8 and amphotericin B against C. albicans 2.2086 are shown in Table 2. In the clinical keratitis experiment, C. albicans at the exponential stage of growth was transferred to the surface of the mouse cornea by corneal subepithelial scarification. After 18 h of inoculation, the formation of eye ulcers with a leathery, tough, raised surface was observed, suggesting that C. albicans had infected the cornea and the keratitis model was successfully established. Furthermore, 48 female animals were randomly treated with four topical eye drop solutions (10 μL): 0.7% saline solution (control), 1 mg/mL of amphotericin B, and 1 mg/mL and 5 mg/mL of W8. The scores of clinical keratitis treatment were evaluated using the clinical standard (Table S1), confirming that the scores of the treatment groups were significantly lower than those of the control group (P 0.05) and can eradicate fungal cells almost up to 80% from eyeballs after treatment compared with that of the control group treated with 0.7% NaCl. Taken together, W8 showed excellent activity against fungi-related keratitis in mice and still maintain relatively higher antimicrobial activity.



2.6 Conformational variability of peptides in different environments CD spectroscopy confirmed the structure conformations of all engineered peptides in different environments. The CD spectroscopy of peptides in 10 mM sodium phosphate buffer (pH 7.4), 30 mM SDS micelles and 50% TFE (trifluoroethanol) solution is shown in Figure 4. The helix-promoting agent TFE was employed to mimic the hydrophobic environments of the bacterial membrane and evaluated the helical propensity of peptides, and the SDS micelles with the negatively charged surface was adopted to simulate an anionic membrane environment23. In 10 mM sodium phosphate buffer, all the peptides adopted randomly unordered conformations based on showing an obviously local minimum dichroic band at approximately 200 nm. In the 50% TFE solution, the CD spectroscopy of all peptides, excluding Kunitzin-RE, OT and P8 exhibited modest α-helical structural propensity via the two minimum dichroic bands at approximately 208 nm and 222 nm. In 30 mM SDS micelles, the spectral characteristic of engineered peptides I8, T8, Y8, A8, F8, L8, W8, and


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TSW-2 showed a maximum dichroic band near 200 nm and a minimum dichroic band at 216-220 nm, indicating a representative β-sheet conformation, while the engineered peptides Kunitzin-RE, OT, P8, G8, N8, D8 and R8 displayed randomly unordered conformations. Dramatically, TSW-1 always showed a consistent α-helical structural characteristic in TFE solution. 2.7 FITC-labeled peptide interaction with the bacterial membrane

To verify the localization of AMPs on the bacterial membrane, superresolution microscopy was employed to directly observe actin localization through the addition of FITC-labeled W8, which can release green fluorescence, and the PI nucleic acid dye was added that can penetrate broken cells and release red fluorescence. Figure 5 shows that the fluorescence emitted by FITC-labeled W8 completely covered the bacterial surface and traced the morphological structure of the bacteria. Furthermore, the fluorescence emitted by the red nucleic acid dye PI was also monitored. Thus, we inferred that bacteria destruction was induced by



the interaction with FITC-labeled W8 and the surface of bacterial membranes, causing compromised membrane integrity. 2.8 Liposome assays In general, liposome preparation was used to simulate different compositions of the cell membrane, such as those in bacterial or mammalian cells. The capacity of the interaction between the phospholipid layer and peptides was measured by the induction of calcein leakage from liposomes23. As shown in Figure 6C, compared with melittin, these results confirmed that W8 exhibited similar superior liposome leakage activity toward E. coli membrane as mimicked by PG/CL/PE (2:1:7), causing more than 40% and 65% leakage at the concentrations of 8 μM and 64 μM, respectively, while Kunitzin-RE showed notably lower than 5% leakage at the highest concentration. Furthermore, for the liposomes formed with PC/cholesterol to mimic the mammalian cell membrane, W8 showed negligible leakage activity with calcein leakage of less than 5%. Conversely, melittin showed higher leakage activity, more than 85% at a


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concentration of 1 μM. Taken together, the above results suggested that W8 failed to display the killing capacity against mammalian cells, demonstrating that it exhibits good cell selectivity, whereas melittin causes severe damage of the mammalian cell membrane. 2.9 LPS binding experiment LPS is a major component on the cell surface of Gram-negative bacteria. Stimulation of LPS led to the production of cellular inflammatory factors and was also the main binding site of AMPs adsorbed onto the surface of bacteria by electrostatic attraction24. Thus, the BODIPY-TR cadaverine was used in a fluorescence-based displacement assay to assess the capacity of the interaction between LPS and peptides. As shown in Figure 6A, in a dose-dependent manner, Kunitzin-RE and melittin presented fluorescence values at approximately 72% and 96%, respectively. However, W8 displayed a better interaction capacity with LPS, corresponding to fluorescence values reaching 99% at 16 μM and 180% at



the highest concentration (64 μM), respectively, suggesting that its LPSbinding capacity was greater than others. 2.10

Outer membrane permeabilization assays Next, the antimicrobial mechanism of W8 was further studied by

exploring the permeabilization of the OM of E. coli 25922 as the model microorganism. Additionally, the typical peptide melittin and Kunitzin-RE were employed as the positive control and negative control, respectively. 1-N-phenylnapthylamine (NPN) was blocked by the cell wall and aqueous conditions; however, once the OM of the cell was permeabilized, NPN was further taken up intracellularly, resulting in the intense fluorescence intensity of NPN in a hydrophobic environment and suggesting the OM integrity was broken25. As shown in Figure 6B, the permeabilization capacity of the OM for these peptides was calculated by the absorbance of hydrophobic the fluorescent probe NPN in a concentration-dependent manner. Kunitzin-RE induced the permeabilization of the OM by only 35% against E. coli 25922 at the highest concentration (64 μM). By contrast,


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more than 50% and 110% of cell membrane permeabilization of W8 at the highest concentration (64 μM) and 4 μM were evaluated, respectively, revealing it was obviously stronger than Kunitzin-RE. Similarly, as the positive control, melittin also displayed stronger permeabilization of the OM, and W8 showed slightly lower membrane permeabilization than melittin. 2.11

Cytoplasmic membrane assay AMPs served to cause the functional expense of the CM integrity,

primarily resulting in perturbations of the electric potential for the CM and direct permeabilization of the CM26. Thus, the membrane potentialsensitive dye-DiSC3-5 was used to assess the disturbance of the bacterial membrane potential interacting with the peptide. Under normal conditions, DiSC3-5 can form nonfluorescent aggregations; however, once the CM was disturbed due to perturbation of the ion flux, DiSC3-5 was taken up into the cytoplasm and formed a monomer with the continued production of fluorescence. As shown in Figure 6D, the influence of the depolarization



of the CM at various concentrations of Kunitzin-RE, W8 and melittin were determined in a dose- and time-dependent manner. W8 displayed a greater depolarization capacity than Kunitzin-RE with a fluorescent intensity (AU) near 3000 at 1×MIC (4 μM), but it was also slightly lower than that induced by melittin with a fluorescent intensity (AU) near 4000 at 1×MIC (2 μM). Next, the permeabilization of the CM with engineered peptides (KunitzinRE, W8 and melittin) was investigated by the release values of cytoplasmic β-galactosidase (Figure 6E). W8 could cause the highest release of cytoplasmic β-galactosidase compared with Kunitzin-RE and melittin. The results showed that W8 exerted a better CM permeabilization capacity and melittin displayed a greater depolarization capacity. 2.12

Morphological observation of bacteria To directly discover and show the integrity of the bacterial membrane

and morphology of the cell, the model bacteria were incubated with peptide treatment at 1×MBC for 1 h, and the membranes of E. coli 25922 and S. aureus 29213 were directly observed by AFM and SEM (Figure 7 A-L).


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For the controls (E. coli 25922 and S. aureus 29213) both presented an intact and smooth membrane surface (Figure 7 A, C, E, I); however, after the incubation of W8 with the bacterial strains, the morphology of the bacterial membrane appeared distinctly abnormal (Figure 7 B, D), including creping and blebbing (Figure 7 F, J) or pore formation (Figure 7 G, K) and content leakage (Figure 7 H, L). Additionally, the intracellular ultrastructural transformation of bacterial cells treated with W8 were further observed by TEM (Figure 7 M-T). For E. coli 25922, the bacterial cells treated with W8 displayed noteworthy separation between the OM and CM (Figure 7 N), and induced pore formation (Figure 7 O), resulting in a large amount of intracellular content leakage, finally leading to clearly visible sparse cytoplasmic distribution and a significant empty area (Figure 7 P) comparable to the control (Figure 7 M). Similarly, compared with the surface of untreated S. aureus 29213 (Figure 7 Q), the surface of W8treated S. aureus 29213, demonstrated a broken OM and the formation of



tiny holes (Figure 7 R, S), further resulting in cytoplasmic leakage sparse distribution of the cytoplasm and intracellular empty areas (Figure 7 T).

3 Discussion

Coinfections of microorganism and multidrug resistant (MDR) strains have been became insurmountable clinical treatment bottlenecks, such as regarding the treatment of catheter site infections, acne and infected skin wounds9, 10. Currently, AMPs, based on their anti-microbial potential and characteristic antibacterial mechanism by generally causing irreversible and overwhelming disruption of bacterial membranes and significantly compromising the production of bacterial resistance, were selected as substitutes for conventional antibiotics as a novel therapeutic candidate27, 28.

Nevertheless, many problems limit the development of AMPs. For

example, naturally occurring peptides extracted from organisms had failed to display higher antimicrobial activity and a wide antibacterial spectrum, instead serving to lyse the membranes of mammalian cells, causing relatively hemolytic or cytotoxic effects and impairing cell selectivity22, 29.


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Additionally, the de novo synthetic peptides produced by the crucial dominant amino acids easily induced poor biocompatibility, thus resulting in the reduction of bacteriostatic potency and potential drug resistance30. Therefore, to keep the inherent advantages and overcome the above disadvantages, currently, simplification of peptide sequences and/or site mutation of amino acids was viewed as an efficient strategy for the optimization of AMPs13, 31. Therefore, it may be a convenient and effective approach to obtain high cell selectivity and broad-spectrum sterilization by the truncation of naturally occurring peptides and residue substitution of the crucial site. Based on previous research results on amphibian AMPs, a series of short peptide variants was designed by truncating and residue substituting Kunitzin-RE, which is released at the skin surface in response to injury and prevents infection by pathogens, and then we investigated the potency of derivative peptides against pathogen bacteria and the effect of changes in the peptide length, positive charge, hydrophobicity and amphiphilicity on



the bioactivity while screening for excellent AMPs with the highest therapy potential20, 22. In this study, to better treat coinfections of microorganism, we expected to screen for excellent derivative peptides containing broadspectrum antimicrobial potency using the antimicrobial assay. First, it was well confirmed that sufficient positive charge is a prerequisite for the electrostatic adsorption of the peptides to the anionic substances of the bacterial membrane surface, and the peptide charge was in a reasonable range, usually +4~+6 served to enhance the antibacterial activity, but the effect of positive charge on the activity had a threshold: excessive positive charge led to the reduction of the antimicrobial potency and cell selectivity32, 33. Furthermore, the side chain guanidinium groups of cationic R residues could contribute to form stronger H-bonds with the phosphorich membrane surface of the bacteria, facilitating electrostatic interaction between the peptide and membrane surface of the bacteria and further enhancing the insertion capacity of the lipophilic region of the peptide


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toward the bacteria membrane, improving the antibacterial potency and broadening the antimicrobial spectrum34, 35. In this study, the results of Table 2 and Table 3 showed that P8 (GM all=39.74 µM) containing four cationic R residues had higher activity than peptides with three cationic R residues such as OT (GM

all=128.00

µM) and D8 (GM

all=128.00

µM);

additionally, R8 (GM gram-positive= 19.03 µM; GM gram-negative= 17.28 µM; GM fungi=

80.63 µM), replaced at the P residue of P8 with the R residue,

broadened the antibacterial spectrum while slightly increasing the antibacterial potency compared with P8 (GM gram-positive= 128.00 µM; GM gram-negative=

16.00 µM; GM fungi= 128.00 µM). Thus, together, these findings

may indicate that arginine and positive charge were the prerequisites for exerting activity, but positive charge indeed showed a threshold such that the further addition of positive charge (from +4 to +5) resulted in no significant efficiency. However, the cationic R residue or positive charge showed a high correlation with the broadening of the antibacterial spectrum



Page 30 of 97

comparable to OT, P8 and R8, implying that positive charge and arginine played a vital role in the bactericidal range. Additionally, the hydrophobicity of the peptide played a decisive role on the bioactivity36. Within a certain range, the hydrophobicity of the peptides was highly correlated with its antibacterial activity, likely explaining why P8 series analogs with different hydrophobicity values by single amino acid substitutions trend toward increased antibacterial activity with increasing hydrophobicity; particularly, W8 (GM gram-positive= 1.19 µM; GM gram-negative= 2.72 µM; GM fungi= 5.04 µM), with the highest hydrophobicity value, displayed the highest antibacterial activity than the other peptides. Compared with the MICs of Kunitzin-RE, W8 obviously reduced the dosage of peptides, with MICs ranging from >128 to 1 mM against

Gram-positive

bacteria

and

significantly

increasing

the

antimicrobial capacity toward bacteria and fungus. Compared with MICs of P8 (GM

gram-positive=

128.00 µM; GM

gram-negative=

16.00 µM; GM

fungi=

128.00 µM) with the same number of cationic residue, W8 dramatically


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increasing the antimicrobial activity and antimicrobial spectrum. Above results showed that hydrophobicity is an important factor affecting the antimicrobial spectrum and antimicrobial activity of AMPs and these results were consistent with the proposed bacteriostatic mechanism of hydrophobic residues that the aggregation of peptides reached a threshold on the surface of the bacterial membrane, and the hydrophobic residues could be inserted into the hydrophobic region of the lipid bilayer of the membrane, resulting in the formation of pore/ion channels on the CM and finally causing cell death37. Collectively, amphipathicity is also an important factor for antimicrobial activity, and many studies have suggested that the hydrophobic and hydrophilic surfaces obtained by the perfect amphipathicity arrangement of amino acids could result in effective bactericidal activity and cell selectivity38. By contrast, other studies have recently shown that perfect amphipathicity AMPs often lead to a simultaneous increase in bactericidal activity and hemolysis while it was



confirmed that appropriate interruption of the arrangement of the hydrophobic and hydrophilic surfaces to convert from perfect amphipathicity to imperfect amphipathicity was a better strategy to obtain a higher antibacterial potential and cell selectivity28, 33, 39, 40. Therefore, to verify the results of previous studies and discuss the relationship between amphipathicity and activity, W8 (GM

all=

2.48 µM) with the highest

antibacterial activity was used as a framework to obtain TSW-1 (GM all= 4.97 µM) and TSW-2 (GM

all=

3.67 µM) with a perfect amphipathic

structure. These results (Table 2 and Table 3) were consistent with studies in which imperfect amphipathicity possessed an advantage on bacteriostatic capacity, and perfect amphipathic peptides did not exhibit better activity but displayed slightly decreased antibacterial activity. The cause may be that imperfect amphipathicity was more conducive to the formation of membrane pores41. Additionally, Our CD results (Figure 4) suggested that these peptides (I8, A8, F8, L8, W8, TSW-1, and TSW-2) presented stronger antibacterial


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potency and exhibited a clear and stable α-helical or β-turn secondary structure, while Kunitzin-RE, OT, P8, N8, D8, and R8, which had relatively weak antibacterial potency, all expressed a disordered structure in SDS solution, further confirming previous research perspectives that the formation of a stable spatial secondary structure was an important factor for the antibacterial activity of AMPs at the molecular structure level33. Based on the demonstration of excellent bactericidal activity, we thought the maintenance of good cell selectivity against erythrocyte cells or eukaryocytes was also an important indicator for considering the clinical application value of AMPs. As shown in Figure 2C, most peptides containing 11 residues showed a poor hemolytic reaction with a hemolytic rate less than 5% compared with the 17-residue Kunitzin-RE peptide with 20% hemolytic rate and melittin with 84% hemolytic rate. Above results confirmed that the peptide chain length could weaken the capacity of cell selectivity42. Nevertheless, many previous studies have suggested that increased cationic character, hydrophobicity and amphipathicity all caused



cytotoxicity and hemolytic reactions33, 43, 44. Thus, we speculated that the effect of chain length on cell selectivity was only a superficial phenomenon because the decrease in the peptide chain length also induced the change in the cationic residues, hydrophobicity and amphipathicity, resulting in obstructed judgment of which parameter accounted for the crucial status of cell selectivity, and it may be implied that the balance of the hydrophobicity and hydrophilicity of the peptide is the most crucial parameter to promote cell selectivity. Additionally, the cytotoxicity results (Figure 2A) were almost consistent with those of our hemolytic rate assay, except for TSW-1 with a cell survival rate of 10.24%, which was inadequate to indicate that a perfect amphipathicity peptide more easily caused cytotoxicity because TSW-2 did not induce cytotoxicity39. Interestingly, the CD results may confirm the above phenomenon that all peptides with a strong antibacterial potency can form a beta-sheet secondary structure in SDS solution, except for TSW-1 with the α-helical secondary structure. Thus, the beta-sheet secondary structure has better cell


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selectivity45. Nevertheless, TSW-1 could cause the production of cytotoxicity, but not hemolytic activity, and may cause cell apoptosis via cell receptor mediation. The salt resistance of AMPs at physiological concentrations is an important guarantee for clinical application (Table 4, 5, 6). It was well known that free ions, such as monovalent cations (Na+ and K+) and divalent cations (Ca2+) in the reaction system, served to hinder the electrostatic attraction of the peptide, further impairing the binging capacity of the peptide against the bacterial membrane surface and resulting in the compromised antimicrobial potency of the peptide46. Peradventure salt ions served to compete with AMPs toward the anionic phosphate groups, such as in LPS or glucan, increasing bacterial membrane rigidity and hindering the pore formation of the bacterial membrane, finally causing decreased antimicrobial potency47, 48. Moreover, the degree of influence on the activity of the peptide depended on the concentration of salt ions21. These adequately explained our findings that the antimicrobial activity of



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most AMPs was significantly affected by salt-free ions including Na+ and Ca2+, which had a higher concentration in the physiological environment but caused slightly or no effect on other free ions with the trace concentration. Interestingly, the antibacterial capacity of W8 not only showed little or no effect on the disruption of most free ions but also facilitated improved antimicrobial potency of the monovalent K+. Some literature sources have suggested that Trp residues presented a bulky indole ring to facilitate the salt resistance of peptides by deeper penetration of the bacterial membrane and increasing the membrane-bound surface area49. Additionally, the latter phenomenon may be explained by monovalent K+ binding to LPS (protecting against bacterial membranes) and then weakening the defensive ability of LPS for the bacterial membrane, finally promoting the antibacterial effect of AMPs 24. Moreover, monovalent K+ may induce self-assembly of the peptide upon altering its conformation to enhance the capacity of binding against the cell membrane48,

50.

Furthermore, TSW-1 was found to show relatively lower salt ion resistance


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than W8 and TSW-2. We speculated it may be that TSW-1 can form an αhelical structure in SDS solution rather than the β-sheet structure of W8 or TSW-2 because the β-sheet structure can maintain good stability of the peptide32. Overall, compared with traditional antibiotic drugs, W8 had a significant antimicrobial spectrum advantage and it was also equivalent in terms of stability. Thus, we initially determined that W8 was the therapeutic agent with the highest potential. However, whether w8 could resist the production of drug-resistant bacteria was the toughest problem. Thus, the drug resistance test (Figure 2D) was performed to evaluate whether bacteria easily developed resistance to the W8. The results showed that w8 was difficult to induce the development of microorganism resistance, and still maintained an effective antibacterial capacity against gentamicin-resistant P. aeruginosa and melittin-resistant P. aeruginosa. Above results demonstrated that W8 had the excellent therapeutic development potential based on its excellent broad and efficient



antimicrobial properties, preferable cell selectivity and its perfect antimicroorganism drug resistance. Additionally, many AMPs displayed stronger antimicrobial activity in vitro but slightly or no activity in vivo. Thus, the stability studies in vivo can more accurately reflect the antimicrobial potential of AMPs in complex physiological environments. Our results qualitatively and quantitatively indicated that W8 served to effectively eliminate most fungal colonies and ideally inhibited the extension of fungal hyphae and infiltration of fungal hyphae into the stroma of the cornea. These results (Figure 3) indicated that W8 maintained a better treatment effect, and, with the peptide concentration increase (1 mg/mL and 5 mg/mL), the bacteriostatic effect was also significantly enhanced, suggesting that the bacteriostatic effect relies on the peptide concentrations. Additionally, W8 (5 mg/mL) showed a similar treatment effect with amphotericin B (1 mg/mL), indicating that W8 could be used as a promising antimicrobial agent in clinical application.


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The replacement of antibiotics with AMPs was expected to follow a novel therapeutic schedule based on the unique bacteriostatic mechanism that hardly induced drug resistance51. Thus, the bacteriostatic mechanism of W8 was further studied to verify the entire interaction between W8 and the cell membrane. Previous studies have shown that the stable secondary structure is a critical factor for the antimicrobial activity of AMPs and some special amino acid types and the arrangement can change the secondary structure tendency of peptides33, 52. The CD results indicated that in 50% TFE solution which mimicked the hydrophobic environment of microbial membrane, all engineered peptides exhibited modest α-helical propensity excluding Kunitzin-RE, OT and P8. The main reason was that KunitzinRE, OT and P8 contained proline, which can destroy the α-helical tendency and caused peptides inactivation when proline occupied at H-H bondinglinked positions60. Meanwhile, in the SDS micelles which mimicked an anionic membrane environment, these engineered peptides (I8, T8, Y8, A8, F8, L8, W8) adopted a stable β-sheet secondary structure, because their



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sequences were formed by the cross arrangement with hydrophobic residues and cationic residues59. In addition, W8 represented a random coil structure in PBS solution; however, it transformed to a stable secondary structure in SDS micelles or TFE solution, confirming that the bacterial membrane environment was essential for the antimicrobial activity of AMPs. Additionally, superresolution microscopy was used to directly observe the FITC-labeled peptide localization against pathogens. As shown in Figure 5, the fluorescence emitted by FITC-labeled peptide completely covered the bacterial surface and traced the morphological structure of the bacteria, while the fluorescence emitted by the red nucleic acid dye PI was also monitored. The above results sufficiently demonstrated that W8 was localized around the bacterial membrane and could exert antibacterial potency by the interaction with the bacterial membrane, causing cell death. Based on the above speculation, the interaction between W8 and different

components

of

the

bacterial


membrane,

including

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lipopolysaccharide (LPS), outer membrane (OM), and cytoplasmic membrane (CM), was further studied to investigate the bacteriostatic action. Thus, the binding activity of W8 to LPS (Figure 6 A) suggested that W8 expressed a dose-dependent increase but displayed a greater binding capacity of LPS with melittin and Kunitzin-RE. It was well known that LPS, which is an anionic component of the outer leaflet on the OM of Gram-negative bacteria, can hinder various host defense molecules to interact with the bacterial membrane by electrostatic attraction24. Thus, we speculated that the combination between AMPs and LPS is the first step toward the interaction of AMPs and the bacterial membrane. Subsequently, the OM permeability test (Figure 6 B) showed that W8 served to present a stronger capacity of OM permeability with an approximately 51% OM permeability efficiency at 1× MIC but approximately 15% at 1/2× MIC, demonstrating that AMPs could accumulate on the surface of the membrane and its concentration needs to reach the threshold to exert a minimum ability of OM permeability. Next, the results of the CM



depolarization level induced by W8 (Figure 6 D) revealed that W8 still exhibited a relatively rapid and strong CM depolarization capacity, corresponding to AUs with approximately 3000 at 1× MIC, but the CM depolarization capacity of W8 was still lower than melittin with a fluorescent intensity (AU) near 4000 at 1×MIC. Meanwhile, the CM permeability test, as measured by the release of cytoplasmic βgalactosidase (Figure 6 E) and LUVs consisting of PG/CL/PE at a 2:1:7 mass ratio (Figure 6 C), which mimic the E. coli membrane, were used to confirm that W8 had excellent CM permeability and can induce the leakage of the intracellular content, resulting in cell death53. Interestingly, the CM permeability capacity of W8 was obviously stronger than melittin and caused more physical damage to the bacterial membrane, this may be the reason why melittin also induced bacterial resistance. Taken together, these results demonstrated that, after W8 penetrated the OM, W8 continued to interact with the CM through potential perturbation of the CM and direct


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mechanical destruction of the CM by the formation of pores and/or channels. Finally, our AFM, SEM, and TEM (Figure 7) studies directly demonstrated that W8 can cause different types of damage to bacterial membranes, such as pronounced blebbing of OM, pore formation of the cell membrane, leakage of the intracellular content induced by bacterial lysis and obvious CM and OM separation, suggesting that W8 has a strong bactericidal effect mainly through the action of the membrane, causing multifaceted damage to the membrane structure. Overall, based on above conclusions, we postulate that W8 induced bacterial cell death through the following process: initially, W8 could combine with LPS by electrostatic adsorption and was attracted to the surface of the membrane. With the aggregation of peptide molecules and reaching a threshold, the hydrophobic core of the peptide was inserted into the phospholipid layer and caused the OM to rupture by the transmembrane electrical potential (the interior/cytosol being negative)47. Consequently, a



mass of peptide molecules continued to interact with the CM, and its hydrophobic core was also inserted into the lipid bilayer of the CM, triggering the potential perturbation and pore/ion channel formation of the CM, causing significant change in the permeability toward the bacterial membrane and a large loss of the intracellular content, resulting in cell death54, 55.

4 Conclusion

In this study, we designed a series of short peptide variants from Kunitzin-RE by simplification and replacement of single amino acids to investigate the effect of chain length, positive charge, hydrophobicity, amphipathicity and secondary structure on the bioactivity. Additionally, W8, with the highest antimicrobial potency, was used as the framework to obtain a set of perfectly amphipathic peptides, to determine the effect of perfect/imperfect amphipathicity on the biological activity. In this system, most short peptide variants exhibited better antimicrobial potency and cell selectivity than Kunitzin-RE, but the influence of the amphipathicity on


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toxicity showed a relatively different result from that in previous studies. Thus, we considered that positive charge and cationic residues (Arg) were prerequisites to promote the activity of AMP within a certain range, but after reaching the threshold, the positive charge and cationic residues (Arg) had little effect on the activity, but positive charge and cationic residues (Arg) showed a relatively high correlation with the broadening of the antimicrobial spectrum. Furthermore, imperfect amphipathicity peptides demonstrated better antimicrobial capacity than perfect amphipathicity peptides, while hydrophobicity was also the most important factor affecting the antimicrobial activity and antimicrobial spectrum of AMPs. Additionally, the effect of the peptide chain length on cell selectivity was only a superficial phenomenon, and an appropriate equilibrium between cationicity and hydrophobicity was the most critical factor toward the improvement of cell selectivity. Moreover, the β-sheet secondary structure may facilitate the improvement of cell selectivity. Among all engineered peptides, W8 had the highest TI value with a rapid sterilization efficiency;



it showed a high salt tolerance in vitro and maintained potent activity in vivo, suggesting that it had excellent clinical stability. W8 had significantly LPS-binding ability and exerted its antibacterial mechanism by targeting the anionic surface of the membrane, damaging the OM/CM integrity and inducing the formation of pore/ion channels, resulting in leakage of the intracellular content and eventually prompting cell lysis. This mechanism may induce the low likelihood of drug resistance development. Undoubtedly, W8 had great potential to replace the clinical status of conventional antibiotics in the treatment of commensalism coinfection of pathogens.

5 Experimental Section

5.1 Synthesis and characterization of peptides The engineered peptides were synthesized by GL Biochem Corporation (Shanghai, China) and were verified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Linear Scientific Inc., USA) to determine their fidelity. The purification


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(>95%) and retention time of the peptide were tested by reverse-phase high-performance liquid chromatography (HPLC) using a GS-120-5-C18BIO column (4.6×250 mm, a 10-μL volume and a nonlinear water/acetonitrile gradient containing 0.1% trifluoroacetic acid at a flow rate of 1.0 mL/min). The quantification of the charge and hydrophobic moment were analyzed online using the HeliQuest analysis website (http://heliquest.ipmc.cnrs.fr/cgi-bin/ComputParamsV2.py). The helical wheel projection was shown online using the helical wheel projection (http://rzlab.ucr.Edu/scripts/wheel/wheel.cgi).

The

three-dimensional

spatial structure projection of the peptide was further predicted online using I-TASSER (http://zhanglab.ccmb.med.umich.edu/I-TASSER/). 5.2 Bacterial strains Escherichia coli (E. coli) ATCC25922, E. coli 078, E. coli K88, E. coli K99, Salmonella pullorum (S. pullorum) ATCC 7913, Salmonella typhimurium (S. typhimurium) ATCC14028 and C7731, Staphylococcus aureus (S. aureus) ATCC 29213, S. aureus ATCC25923, Staphylococcus



epidermidis (S. epidermidis) ATCC12228, Pseudomonas aeruginosa (P. aeruginosa) ATCC 27853 and methicillin-resistant S. aureus ATCC 43300 were obtained from the College of Veterinary Medicine, Northeast Agricultural University (Harbin, China), and E. coli UB1005 was kindly obtained from the State Key Laboratory of Microbial Technology (Shandong University, China). C. albicans cgmcc2.2086, C. parapsilosis cgmcc2.3989 and C. tropicalis cgmcc2.1975 were purchased from the China General Microbiological Culture Collection Center (CGMCC, Beijing, China). 5.3 Antimicrobial activity assays The antibacterial potency of all peptides was tested using a method adopted from the National Committee for Clinical Laboratory Standards (NCCLS) with slight modifications56. The special method for these experiments was previously described. Bacterial cells were grown to the mid-logarithmic phase and diluted in MHB to reach a final concentration of (0.5–1) × 105 CFU mL−1. Consequently, the peptides were serially


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diluted in 0.2% BSA solution while mixed with equal volumes of bacterial solution in a 96-well plate. After incubation for 24 h at 37 °C, the minimum inhibitory concentrations (MICs) were measured using a microplate reader with the absorbance at 492 nm as the lowest peptide concentration that inhibited 95% of the bacterial growth. Furthermore, 50 μL of each incubation mixture was further transferred to agar plates, followed by incubating overnight to verify the minimum bactericidal concentration (MBC) at the lowest peptide concentration that can kill greater than 99.9% of the bacterial cells. These results were from at least three independent experiments and each independent experiment contained three technical replicates. Similarly, the antifungal activity was also detected via a standardized broth microdilution method (Clinical and Laboratory Standards Institute (CLSI) document M27-A2) with modifications57. Briefly, yeast colonies were selected and diluted in RPMI 1640 growth medium buffered with morpholinepropanesulfonic acid (MOPS) and were adjusted to the



terminal concentration of (0.5–1) × 103 CFU mL−1. Subsequently, the peptides were serially diluted in 0.2% BSA solution while being mixed with equal volumes of fungal solution in 96-well plates. After incubation with 48 h at 28 °C, the minimum inhibitory concentration (MIC) of the peptides was measured via the absorbance at 492 nm using a microplate reader with the lowest peptide concentration that inhibited 95% of the fungal growth. Additionally, 50-μL samples from each well was plated on YM agar plates, followed by incubation for 48 h at 28 °C. Furthermore, we determined the minimum fungicidal concentration (MFC)—that is, the lowest peptide concentration that can kill at least 99.9% of the fungal cells. Finally, at least three independent experiments were conducted for the assay and each independent experiment was performed using three technical replicates. The kinetics of the sterilization activity of the peptide was further evaluated. E. coli ATCC 25922, S. aureus ATCC 29213 and C. albicans cgmcc 2.2086 (at a final concentration of 0.5-1×105 CFUmL-1) were


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treated with peptides at a 1×MBC/MFC concentration, and aliquots of the mixture were collected at different times to calculate the cell survival rate. An untreated control group was also included. The results of kinetic were the mean values of three independent experiments and each independent experiment contained three technical replicates. Finally, P. aeruginosa ATCC 27853 was selected as the model of drug resistance study. Overnight cultures of P. aeruginosa cell serially passaged by 100-fold dilution in 2 mL batch cultures every 24 h in MHB that contained the sub-MBC concentration of the drugs. The MBC values of the drugs against every one passage’s cells were tested. As a control, MBCs were also tested using cells that were serially passaged in fresh MHB without drugs. The assay was conducted independently in triplicate with three biological replicates. 5.4 Biocompatibility assays The murine macrophage cell line RAW264.7 was used to determine the cytotoxicity of the peptides by the MTT assay, and healthy human red



blood cells (hRBCs) were used to evaluate the hemolytic rates of the peptides as previously described33. Briefly, for the MTT assay, 1.0-2.0×104 cells/well were inoculated into 96-well plates and were interacted with various peptides (2-64 µM) for 18-24 h at 37°C in 5% CO2. Subsequently, the cell culture was further incubated with MTT (0.5 mg/mL) for 4 h at 37°C. The supernatant was discarded, and the formazan crystals were dissolved in 150 µL of DMSO while the solution was further measured using a microplate reader (TECAN GENios F129004; TECAN, Austria) at OD 570 nm. A healthy donor (Zhanyi Yang, Harbin, China) provided a 1-mL sample of fresh hRBCs that was collected and resuspended in PBS (pH 7.4) to obtain a dilution solution of ~1% (v/v) erythrocytes. Next, the mixtures of equal volumes of hRBC solution and the peptides at different concentrations (1-128 µM) were incubated for 1 h at 37°C. The mixtures were centrifuged (1000×g, 10 min), and then the supernatants were transferred to a new 96-well plate to assess the release of hemoglobin at


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576 nm using a microplate reader (TECAN GENios F129004; TECAN, Austria). Additionally, untreated hRBCs served as the negative control (0% hemolysis) and 0.1% Triton X-100 treatment served as the positive control (100% hemolysis). The peptide concentration that caused a hemolytic rate >10% is considered the minimal hemolysis concentration (MHC). The percent hemolysis was calculated via the following formula: Percent hemolysis = [(A − A0) / (At − A0)] ×100 where A represents the absorbance of the peptide sample at 576 nm, and A0 and At represent 0% and 100% hemolysis, determined in 10 mM PBS and 0.1% Triton X-100, respectively. At least three independent experiments were conducted for the biocompatibility assays and three technical replicates were used in each independent experiment.

5.5 Salt tolerance assays The salt sensitivity assay was used to analyze the salt tolerance capacity of all peptides. E. coli ATCC 25922, S. aureus ATCC 29213 and C.



albicans cgmcc 2.2086 were incubated in the presence of different terminal concentrations of physiological salts (150 mM NaCl, 4.5 mM KCl, 6 μM NH4Cl, 8 μM ZnCl2, 1 mM MgCl2, 2 mM CaCl2 and 4 μM FeCl3) based on our previous protocol28. The MIC was determined as described above, and the results were from three independent assays and each independent experiment was performed using three technical replicates. 5.6 In vivo mouse keratitis treatment model 5.6.1 Source of mice Animal care and treatment complied with the standards described in the Guidelines for the Care and Use of Laboratory Animals of the Northeast Agricultural University (NEAU-[2011]-9) to Publication of Chemical Research. 48 female C57BL/6 mice (6-8 weeks old, weighing 20.29 ± 1.75 g) were purchased from Liaoning Changsheng Biotechnology Co. Ltd (Shenyang, China) with twelve female animals per group. Moreover, all mice were given a one-week to acclimatization.

5.6.2 Establishment of scratch keratomycosis model


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The C. albicans colonies were picked in YMB and were incubated in a shaker at 22 °C and 100 rpm until the concentration of the bacteria reached the mid-log phase. Next, the bacterial solution was centrifuged (1,000 × g, 10 min) and washed three times, and then the bacterial cells (OD600 = 0.6) were resuspended in physiological saline to prepare for use. In total, 48 mice were used in this study. The mouse fungal keratomycosis model was established using the above bacterial liquid as eye drops (20 μL), and the specific steps to establishing a fungal keratitis model were as follows. First, the mice were anesthetized by sodium pentobarbital (70 mg/kg) (Aladdin, Shanghai) via intraperitoneal injection (I.P.). An additional topical anesthetic in the form of 0.5% tetracaine hydrochloride eye drops (Aladdin, Shanghai) was also administered. Next, after the mice were anesthetized, a 9-gauge needle was used to gently scratch the corneal center to cause the corneal epithelium to be traumatically removed. Thereafter, the eyelids were gently pulled and bacterial solution (20 μL, OD600= 0.6) was added on the scratches. Finally,



after the C. albicans cells were grown and inoculated onto the eyeball for 18 h, an eye ulcer with a leathery, tough, raised surface was observed, confirming that the scratch keratomycosis model was successfully established. A disease grade from 0 (no disease) to 4 (severe disease) was established to evaluate the treatment efficacy (Table S1). Additionally, all the mice were immune suppressed by subcutaneous injection of cyclophosphamide (100 mg/kg) (Aladdin, Shanghai). 5.6.3 Evaluation of the keratitis treatment efficacy using the peptides The animals were randomly divided into four groups of 12 female mice each and the four groups were treated with 4 topical eye drop solutions: physiological saline (control), 1000 mg/L of amphotericin B (Sigma), and 5000 mg/L and 1000 mg/L of W8. The eye drops (20 μL each) were administered by dripping onto the eyeball of the mice every 5 min during the first hour, and then every 30 min during the next 7 h. All the mice were sacrificed after the execution of the last eye drop, and all the eyeballs treated with the eye drop solutions were collected immediately. Six


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eyeballs from each group were collected for histology, and the remaining 6 eyeballs were homogenized for quantitative fungal recovery study. The fixed eyeballs were embedded in paraffin, sectioned and stained with Periodic acid Schiff (PAS) using standard protocols. 5.7 Antimicrobial mechanism studies 5.7.1 Localization of FITC-labeled peptides To further confirm the localization of the peptide targets, E. coli ATCC 25922 and S. aureus ATCC 29213 were incubated with FITC-labeled peptides and propidium iodide (PI), a dye that stains nucleic acids in cells when the membrane integrity is compromised; subsequently, they were directly observed by superresolution microscopy and a Deltavision OMX system26. Microbial cells (OD600=0.2) were incubated with FITC-labeled peptides at a 1×MBC concentration at 37°C for 30 min. Next, the samples were centrifuged (1,000×g, 10 min) and washed three times and then were resuspended with PI (10 μg mL−1) in PBS buffer. After incubation for 15 min at 4 °C, the unbound PI dye was discarded by washing with an excess



of PBS buffer. The samples were smeared on the glass slide and observed using the Deltavision OMX system with 488 and 535 nm bandpass filters for FITC and PI excitation, respectively. Moreover, cells without peptides served as controls. The graph results from three independent scans with three technical replicates. 5.7.2 CD measurements The CD spectra (λ190−250 nm) of the peptides (35 μM) were expressed in 10 mM PBS, 30 mM sodium dodecyl sulfate (SDS) micelles and 50% trifluoroethyl alcohol (TFE) using a J-820 spectropolarimeter (Jasco, Tokyo, Japan) using a quartz cell with a 1.0-mm path length. We performed three independent scans and an average of >3 runs was made to obtain at least three technical replicates for each sample. The obtained CD spectra were then converted to the mean residue ellipticity using the following equation: θM = (θobs .1000)/ (c.l.n).


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where θM indicates the residue ellipticity (deg cm2 dmol−1), θobs represents the observed ellipticity corrected for the buffer at a given wavelength (mdeg), c is the peptide concentration (mM), l is the path length (mm), and n is the number of amino acids. 5.7.3 LPS binding assay The binding affinity between the peptide and LPS was determined using the BODIPY-TR-cadaverine (BC Sigma, USA) displacement assay58, in which a probe bound to cell-free LPS is self-quenched but fluoresces when released in solution. LPS from E. coli O111:B4 (50 μg/mL) was incubated with BODIPY-TR-cadaverine (5 μg/mL) in Tris buffer (50 mM, pH 7.4) for 4 h. Next, equal volumes of LPS-probe solution and the peptides of various concentrations (1-64 µM) were incubated in a sterile 96-well black plate for 1 h at 37°C, and the fluorescence was measured using a spectrofluorophotometer (Infinite 200 pro; Tecan, China) at excitation and emission wavelengths of 580 nm and 620 nm, respectively; the results were from three independent experiments and each independent experiment



contained three technical replicates. The values were transformed to %ΔF (AU) using the following equation: %ΔF (AU) = [(Fobs - F0) / (F100-F0)] ×100. where Fobs is the observed fluorescence at an obtained peptide concentration, F0 is the initial fluorescence of BC with LPS without the addition of peptides, and F100 is the BC fluorescence with LPS upon the addition of 10 μg mL-1 polymyxin B, a prototype LPS binder viewed as the positive control. 5.7.4 Outer membrane permeabilization Mid-log phase E. coli ATCC 25922 cells (OD600 = 0.2) were incubated with NPN (10 μM) in 5 mM HEPES buffer (pH 7.4, containing 5 mM glucose) for 30 min, and background fluorescence was recorded for subtraction (excitation λ=350 nm; emission λ=420 nm) using an F-4500 fluorescence spectrophotometer (Hitachi, Japan). Subsequently, the 100μL cell suspension extract was mixed with equal volumes of peptide solution (1 to 64 μM) in a sterile 96-well black plate. The fluorescence was


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always recorded until no further fluorescence increase was detectable. The results were transformed to the percent NPN uptake using the equation: NPN uptake (%) = (Fobs − F0)/ (F100 − F0) × 100%. where Fobs is the observed fluorescence at an additive peptide concentration, F0 is the initial fluorescence of NPN with microbial cells in the absence of peptide, and F100 is the fluorescence of NPN with microbial cells upon addition of 10 μg/mL of polymyxin B (Sigma) (for bacteria)/0.1% Triton X-100 as a positive control. The results were from three independent experiments and each independent experiment was performed by using three technical replicates. 5.7.5 Liposome leakage assay Calcein-entrapped large unilamellar vesicles (LUVs) comprising PG/CL/PE (2:1:7, w/w/w, mimicking the E. coli membrane57) and PC/cholesterol (10:1, w/w, mimicking the human erythrocyte cell membrane57) were prepared as described previously. Each phospholipid was dissolved in chloroform at each of the above narrative ratios while



dried with nitrogen and then was resuspended in calcein solution (70 mM calcein, 10 mM Tris, 150 mM NaCl, 0.1 mM EDTA, pH 7.4). The suspension was frozen and thawed 20 times in liquid nitrogen and was continuously squeezed 20 times using a polycarbonate filter (two stacked 100-nm pore size filters) using a LiposoFast extruder (Avestin, Inc., Canada). The free calcein was removed by gel filtration using a Sephadex G-50 column. The lipid concentration was measured by the method described by Stewart. LUVs were incubated (100 μM) with the peptides at concentrations that ranged from 1 to 64 μM for 15 min. The dye released from an LUV was calculated by measuring the fluorescence intensity with an excitation wavelength of 490 nm and an emission wavelength of 520 nm using a spectrofluorophotometer (the Infinite 200 pro; Tecan, China). A 0.1% Triton X-100 solution was used to determine 100% dye leakage as the positive control. The percentage of dye leakage by the peptide was calculated using the following formula: Dye release (%) = (Fobs - F0) / (F100 - F0) ×100%.


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where F0 is the fluorescence intensity of liposomes (background), and Fobs and F100 are the respective intensities of the fluorescence of the peptide and Triton X-100. The assay was conducted independently in triplicate and each independent experiment was performed with three biological replicates.

5.7.6 CM depolarization assay

Mid-log phase E. coli ATCC 25922 cells (OD600= 0.05) were incubated with 0.4 μM DiSC3-5 and 100 mM K+ in 5 mM HEPES buffer (pH 7.4, containing 20 mM glucose) until the fluorescence was detected to achieve a stable reduction. The background fluorescence was recorded (excitation λ = 622 nm, emission λ = 670 nm) using an F-4500 fluorescence spectrophotometer (Hitachi, Japan). Next, 2 mL of cell suspension was added to a 1-cm quartz cuvette and was mixed with various concentrations of peptides. Changes in the fluorescence were recorded from 0 to 3000 s. The assay was conducted independently in triplicate with three biological replicates.



5.7.7 Inner membrane permeability assay E. coli 25922 cells were incubated to the mid-log phase in MHB containing 2% lactose at 37°C and then were obtained and diluted to an OD600 =0.05 in 5 mM HEPES buffer (pH 7.4, containing 20 mM glucose and 1.5 mM ONPG). Subsequently, equal volumes of cell suspension and the peptide solution (1 to 64 μM) were added to a sterile 96-well plate and were incubated at 37°C. The OD420 measurements of the mixture were recorded every 6 min from 0 to 120 min, reflecting ONPG influx into the cells while analyzing the peptide-caused permeabilization function of the inner membrane. The assay was conducted independently in triplicate with three biological replicates. 5.7.8 Morphological observation of microorganisms For the SEM sample preparation, mid-log-phase E. coli ATCC 25922 cells and S. aureus ATCC 29213 cells (OD600= 0.02) were treated with the peptides at 1 × MBC for 1 h, and then were collected and fixed with 2.5% (w/v) glutaraldehyde at 4°C overnight. Next, the samples were


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continuously treated with a graded ethanol series (50%, 70%, 90%, and 100%) for 10 min, 100% ethanol for 15 min, a mixture (v: v=1:1) of 100% ethanol and tert-butanol for 15 min and finally with absolute tert-butanol for 15 min. The samples were dehydrated in a critical point dryer with liquid CO2, coated with gold-palladium, and observed using a Hitachi S4800 SEM. For AFM sample preparation, the microbial suspension tested with peptides was initially prepared as that for SEM. Next, an extract of 10 μL of microbial suspension was smeared onto the glass slide, followed by air drying. The image results were obtained using a Bioscope atomic force microscope (Bruker, USA). The TEM samples were initially prepared as that for SEM. After prefixation with 2.5% glutaraldehyde at 4°C overnight, the samples were postfixed with 2% osmium tetroxide for 70 min and washed twice with PBS (pH 7.2). Subsequently, the samples were continuously treated with a graded ethanol series (50%, 70%, 90%, and 100%) for 8 min, 100% ethanol



for 10 min, a mixture (v: v =1:1) of 100% ethanol and acetone for 15 min and absolute acetone for 15 min. The samples were then immersed in 1:1 mixtures of absolute acetone and epoxy resin for 30 min and pure epoxy resin overnight. Next, the samples were sectioned using an ultramicrotome, stained with uranyl acetate and lead citrate, and observed using a Hitachi H-7650 TEM. Above all electron microscope observations were performed with three independent scans with three replicates. 5.7.9 Statistical Analysis Statistical analysis was performed using a one-way classification of ANOVA and Student’s t-test (two-tailed). The data were analyzed using Statistical Package for the Social Sciences (SPSS) version 25.0 (Chicago, IL, USA). Quantitative data are expressed as means ± standard deviation (SD), and P < 0.01 was considered as statistically significant. ANCILLARY INFORMATION AUTHOR INFORMATION Corresponding Author Information:


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*E-mail: [email protected]. Tel: +86 451 55190685. Fax: +86 451 55103336. Author contributions Z.Y.Y and A.S.S designed and conceived the experiments; Z.Y.Y and S.Q.H conducted the main experiments assay; J.J.W and Y.Y conducted the in vivo assay. S.Q.H and Y.B.L conducted the SEM/TEM assay. Z.Y.Y wrote the main manuscript text. L.C.Z and A.S.S. supervised the work and revised the final version of the manuscript. All of authors have read and approved the final version of the manuscript. Notes Competing financial interests: The authors declare no competing financial interests. ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (Grants 31472104, 31672434), the China Agriculture Research



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System (CARS-35) and the Program for Universities in Heilongjiang Province (1254CGZH22). ABBREVIATIONS USED AMP, antimicrobial peptide; hRBC, human red blood cell; LPS, lipopolysaccharide; BC, BODIPY-TR-cadaverine; SDS, sodium dodecyl sulfate; TFE, trifluoroethyl alcohol; GM, geometric mean; TI, therapeutic index; RP-HPLC, reverse-phase high-performance liquid chromatography; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; CD, circular dichroism; SEM, scanning electron microscopy; AFM, atomic force microscopy; TEM, transmission electron microscopy; PI, propidium iodide; MALDI-TOF MS, matrix-assisted laser desorption/ionization time of flight mass spectrometry; MOPS, morpholinepropanesulfonic acid; LUV,

large

unilamellar

vesicle;

PC,

phosphatidylcholine;

PG,

phosphatidylglycerol; PE, phosphate dylethanolamine; CL, cholesterol, cardiolipin; MBC, minimum bactericidal concentration; MFC, minimum fungicidal concentration; SSIs, surgical site infections; HAI, healthcare-


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associated infection; PAS, Periodic Acid-Schiff stain; NPN, 1-Nphenylnapthylamine; DiSC3-5, 3,3-Dipropylthiadicarbocyanine iodide; ONPG, o-Nitrophenyl-β-D-Galactopyranoside; MHC, minimal hemolytic concentration; OM, outer membrane; CM, cytoplasmic membrane.

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Table 1. Peptide design and their key physicochemical parameters Compound

peptide

sequence

theoretical MWa

measured MW

net charge

Hb

µHrelc

1

Kunitzin-RE

AAKIILNPKFRCKAAFC-NH2

1894.36

1891.39

4

0.549

0.225

95.58

2

OT

AAKIILNPKFR-NH2

1270.57

1269.61

3

0.440

0.174

98.78

3

P8

AARIILRPRFR-NH2

1368.68

1367.72

4

0.399

0.211

98.02

4

I8

AARIILRIRFR-NH2

1384.72

1383.75

4

0.497

0.250

98.14

5

G8

AARIILRGRFR-NH2

1328.61

1327.65

4

0.334

0.208

96.74

6

N8

AARIILRNRFR-NH2

1385.66

1384.70

4

0.279

0.220

98.33

7

T8

AARIILRTRFR-NH2

1372.67

1371.71

4

0.357

0.206

97.83

8

Y8

AARIILRYRFR-NH2

1434.73

1433.78

4

0.421

0.216

95.16

9

A8

AARIILRARFR-NH2

1342.64

1341.68

4

0.362

0.206

95.46

10

F8

AARIILRFRFR-NH2

1418.74

1417.78

4

0.496

0.250

97.24

11

L8

AARIILRLRFR-NH2

1384.72

1383.77

4

0.488

0.245

96.29

12

D8

AARIILRDRFR-NH2

1386.65

1385.69

3

0.264

0.226

97.97

13

R8

AARIILRRRFR-NH2

1427.75

1426.79

5

0.242

0.236

97.56

14

W8

AARIILRWRFR-NH2

1454.77

1456.81

4

0.538

0.275

99.06

15

TSW-1

AARRILRWIFR-NH2

1454.77

1456.82

4

0.538

0.652

97.57

16

TSW-2

AARIILRWFRR-NH2

1454.77

1456.82

4

0.538

0.662

97.08

number

a

Purity (%)

Molecular weight (MW) was confirmed by mass spectroscopy (MS). b Hydrophobicity (H) values means the total hydrophobicity (sum of all residue

hydrophobicity indices) divided by number of residues and were calculated from http://heliquest.ipmc.cnrs.fr/cgi-bin/ComputParams.py. c Relative hydrophobic moment (μHrel) values were employed to analyze the level of amphipathicity of all peptides and were calculated from http://heliquest.ipmc.cnrs.fr/cgi-bin/ComputParams.py.


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Table 2. MICsa (MBCsb) (MFCd) (μM) of the engineered peptides against bacteria strains and fungal strains

Gram-negative bacteria

Peptite

E. coli

E. coli

25922

1005

S.

S.

Pseudomonas

typhimurium

pullorum

aeruginosa

14028

7913

27853

Gram-positive bacteria

E. coli

E. coli

E. coli

K88

K99

078

S. typhimurium C77-31

MRSA (c) 43300

S. epidermidis 12228

S. aureus 25923

Fungi

S. aureus 29213

C. albicans

C. parapsilosis

C. tropicalis

cgmcc

cgmcc2.3989

cgmcc2.1975

2.2086

Kunitzin-RE

32(64)

64(128)

64(128)

64(>128)

>128

32(64)

32(64)

>128

>128

>128

>128

64(>128)

>128

>128

>128

>128

OT

>128

>128

>128

>128

>128

>128

>128

>128

>128

>128

>128

>128

>128

>128

>128

>128

P8

16(32)

8(16)

16(32)

16(64)

>128

16(32)

16(32)

8(16)

16(32)

>128

>128

>128

>128

>128

>128

>128

I8

4(4)

4(8)

2(4)

2(4)

16(32)

2(4)

4(4)

2(4)

4(8)

2(4)

2(4)

2(4)

2(4)

8(8)

4(8)

4(4) 16(32)

G8

4(8)

4(8)

4(8)

4(8)

16(32)

2(4)

2(8)

4(8)

8(160

8(8)

8(16)

16(32)

8(8)

128

64

N8

32(64)

16(32)

32(64)

16(32)

>128

32(64)

16(32)

16(32)

32(128)

>128

>128

>128

>128

>128

>128

>128

T8

8(16)

4(8)

16(32)

8(16)

>128

4(8)

4(8)

4(8)

16(32)

16(32)

16(32)

16(32)

16(32)

128

64

32(64)

Y8

8(16)

4(8)

8(16)

4(8)

16(64)

4(8)

4(8)

4(4)

4(8)

4(8)

2(4)

8(16)

4(8)

32(64)

64(128)

16(32)

A8

4(4)

2(4)

4(4)

4(8)

16(32)

2(4)

2(4)

4(4)

4(8)

2(4)

2(4)

4(8)

4(8)

16(32)

16(16)

4(4)

F8

8(8)

4(8)

4(8)

4(8)

>128

4(8)

2(4)

4(8)

4(8)

2(4)

2(4)

2(4)

2(4)

8(16)

4(8)

4(4)

L8

4(8)

4(8)

4(8)

4(8)

8(32)

4(8)

2(4)

4(8)

4(8)

2(4)

2(4)

2(4)

2(4)

8(16)

4(8)

4(4)

D8

>128

>128

>128

>128

>128

>128

>128

>128

>128

>128

>128

>128

>128

>128

>128

>128

R8

8(16)

16(64)

32(64)

16(32)

>128

16(32)

8(16)

16(32)

8(16)

16(64)

16(32)

32(64)

16(32)

128

64(128)

64(128)

W8

4(4)

4(4)

2(4)

2(4)

4(4)

2(4)

2(4)

2(2)

4(4)

1(1)

1(4)

2(4)

1(2)

8(8)

4(8)

4(4)

TSW-1

4(4)

4(4)

4(8)

4(8)

8(16)

4(8)

4(8)

4(4)

4(8)

8(8)

4(8)

8(8)

4(8)

8(8)

8(16)

4(4)

TSW-2

2(2)

2(4)

4(8)

4(8)

8(16)

2(4)

2(4)

2(4)

4(8)

8(8)

2(4)

8(8)

4(8)

8(8)

4(8)

4(4)

Melittin

2(4)

2(2)

2(2)

2(2)

2(4)

1(2)

2(2)

2(2)

2(4)

1(4)

2(4)

8(8)

4(8)

4(8)

2(4)

4(4)

Polymyxin B

2(4)

1(2)

2(2)

1(1)

2(4)

0.5(1)

1(1)

1(1)

2(4)

64(128)

32(64)

64(>128)

32(64)

32(64)

8(16)

>128

Gentamicin

1(1)

0.5(1)

2(4)

1(2)

1(1)

1(1)

0.5(0.5)

0.5(0.5)

1(2)

8(16)

1(2)

1(1)

1(1)

>128

>128

>128

Ceftazidime

2(2)

1(2)

1(1)

1(2)

2(4)

1(1)

0.5(1)

0.5(0.5)

1(1)

16(32)

16(32)

16(32)

16(16)

>128

>128

>128

NTe

NT

NT

NT

NT

NT

NT

NT

NT

NT

NT

NT

NT

1(2)

2(4)

1(2)

Amphotericin B

Minimum inhibitory concentration (MIC, μM) was determined as the lowest concentration of peptide that inhibited 95% of the bacterial growth. Data are representative of three independent experiments. bMinimum bactericidal concentrations (MBC,

a

μM) were determined as the lowest peptide concentration that killed greater than 99.9% of the bacterial cells. The data were derived from representative value of three independent experimental trials. cMethicillin-resistant S. aureus. dMinimum fungicidal concentrations (MFC) were determined as the lowest peptide concentration that killed at least 99.9% of the fungal cells. The data were derived from representative value of three independent experimental trials. eNT was no tested.



Page 86 of 97

Table 3. MHC, GM, and TI values of the engineered peptides GMb Gram-negative

Gram-positive

bacteria

bacteria

32

64.00

OT

>128

P8

>128

I8

128

G8

>128

4.32

9.51

N8

>128

27.43

128.00

T8

>128

9.33

16.00

Y8

>128

5.44

A8

>128

3.70

F8

>128

L8

TIc Gram-negative

Gram-positive

bacteria

bacteria

0.50

128.00 39.74 3.22

50.80 128.00 64.00

4.00 2.83

5.88

>128

D8

peptide

Fungi

All

0.30

0.25

0.39

2.00

2.00

2.00

2.00

16.00

2.00

2.00

6.44

37.33

64.00

25.40

39.74

8.35

54.26

26.91

5.04

30.64

53.82

9.33

2.00

2.00

4.76

15.32

27.43

16.00

4.00

16.71

32.00

7.03

47.03

64.00

8.00

36.44

10.08

4.18

69.12

90.51

25.40

61.29

2.00

5.04

4.36

43.55

128.00

50.79

58.69

4.00

2.00

5.04

3.51

64.00

128.00

50.79

72.88

>128

128.00

128.00

128.00

128.00

2.00

2.00

2.00

2.00

R8

>128

17.28

19.03

80.63

23.63

14.81

13.45

3.175

10.83

KunitzinRE

MHCa

Fungi

All

107.63

128.00

83.00

128.00

128.00

128.00

16.00

128.00

128.00

3.43

2.00

5.04

W8

>128

2.72

1.19

5.04

2.48

94.06

215.30

50.79

103.10

TSW-1

>128

4.32

5.66

6.35

4.97

59.26

45.25

40.32

51.54

TSW-2

>128

2.94

4.76

5.04

3.67

87.09

53.82

50.79

69.79

Melittin

1

1.85

2.83

3.17

2.28

0.54

0.35

0.32

0.44

aMHC

is the minimum hemolytic concentration that caused 10% hemolysis of human red blood cells. Data are representative of three

independent experiments. When no detectable hemolytic activity was observed at 128 μM, a value of 256 μM was used to calculate the therapeutic index. bThe geometric mean (GM) of the peptide MICs against bacteria and fungi was calculated. When no detectable antimicrobial activity was observed at 64μM, a value of 128 μM was used to calculate the therapeutic index. cTI is calculated as MHC/GM. Larger values indicate greater cell selectivity.


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Table 4. MIC values of peptides against E. coil ATCC 25922 in the presence of physiological salts. control

NaCla

KCla

CaCl2a

Kunitzin-RE

32

OT

>128

>128

32

>128

64

64

32

32

>128

128

>128

>128

>128

>128

>128

P8 I8

16

128

16

128

64

32

16

16

4

8

2

32

16

4

4

4

G8

4

64

8

64

16

4

4

8

N8

32

128

64

>128

128

32

32

16

Peptide

MgCl2a

ZnCl2a

FeCl3a

NH4Cla

E. coil ATCC 25922

T8

8

>128

32

>128

128

8

16

8

Y8

8

>128

32

>128

64

8

16

8

A8

4

32

8

32

16

4

4

2

F8

8

64

16

32

32

4

16

4

L8

4

64

8

32

16

4

8

4

D8

128

>128

128

>128

>128

>128

>128

>128

R8

8

128

64

128

64

8

32

8

W8

4

8

2

4

4

4

4

4

TSW-1

4

64

8

16

8

8

8

4

TSW-2

2

8

2

4

2

2

2

2

Melittin

2

4

2

8

4

2

2

2

Polymyxin B

2

4

2

16

1

2

2

2

Gentamicin

1

4

1

1

1

1

1

1

Ceftazidime

2

4

2

2

2

2

2

2

aThe

final concentrations of NaCl, KCl, NH4Cl, MgCl2, CaCl2, ZnCl2, and FeCl3 were 150 mM, 4.5 mM, 6 μM, 1 mM, 2 mM, 8 μM, and 4

μM, respectively, and the control MIC values were determined in the absence of these physiological salts. The data were derived from representative value of three independent experimental trials.

Table 5. MIC values of peptides against S. aureus ATCC 29213 in the presence of physiological salts. control

NaCla

KCla

CaCl2a

MgCl2a

ZnCl2a

FeCl3a

NH4Cla

Kunitzin-RE

>128

>128

>128

>128

>128

>128

>128

>128

OT

>128

>128

>128

>128

>128

>128

>128

>128

P8

>128

>128

>128

>128

>128

>128

>128

>128

I8

2

8

2

16

2

2

2

2

G8

8

>128

8

64

16

8

8

16

N8

>128

>128

>128

>128

>128

>128

>128

>128

T8

16

>128

32

64

32

16

16

16

Y8

4

>128

8

64

8

2

4

4

A8

4

>128

8

16

8

4

4

4

F8

2

32

2

4

2

2

4

2

L8

2

16

2

4

2

2

4

2

D8

>128

>128

>128

>128

>128

>128

>128

>128

Peptide S. aureus ATCC 29213

R8

16

128

32

128

32

16

16

16

W8

1

4

1

2

2

1

1

2

TSW-1

4

64

4

8

8

4

4

8

TSW-2

4

32

4

8

4

2

4

4

Melittin

4

8

4

8

4

4

4

4

Polymyxin B

32

64

32

64

32

32

32

32

Gentamicin

1

2

1

4

1

1

1

1

Ceftazidime

16

16

16

32

16

16

16

16

aThe





Page 88 of 97

Table 6. MIC values of peptides against C. albicans cgmcc2.2086 in the presence of physiological salts. control

NaCla

KCla

CaCl2a

MgCl2a

ZnCl2a

FeCl3a

NH4Cla

Kunitzin-RE

>128

>128

>128

>128

>128

>128

>128

>128

OT

>128

>128

>128

>128

>128

>128

>128

>128

P8

>128

>128

>128

>128

>128

>128

>128

>128

I8

8

32

8

64

16

8

8

8

G8

128

>128

128

>128

>128

>128

128

>128

N8

>128

>128

>128

>128

>128

>128

>128

>128

T8

128

>128

>128

>128

>128

>128

>128

>128

Y8

32

128

64

128

64

64

64

64

A8

16

64

16

64

32

16

16

16

F8

8

32

8

64

16

8

8

4

L8

8

32

8

32

8

8

8

8

D8

>128

>128

>128

>128

>128

>128

>128

>128

Peptide C. albicans cgmcc2.2086

R8

128

>128

128

>128

>128

>128

>128

>128

W8

8

16

8

32

16

8

8

8

TSW-1

8

32

16

128

16

8

8

8

TSW-2

8

16

8

32

16

8

8

8

Melittin

4

8

4

8

4

4

4

4

Polymyxin B

32

128

32

>128

32

32

32

32

Gentamicin

>128

>128

>128

>128

>128

>128

>128

>128

Ceftazidime

>128

>128

>128

>128

>128

>128

>128

>128

aThe




Page 89 of 97 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Kunitzin-RE

OT

P8

W4

TSW-1

TSW-2

Figure 1. The helical wheel projections of peptides. Among these helical wheel projections, by default the output presents the charged residues are blue, hydrophobicity residues are yellow, uncharged residues are light pink, alanine and proline are gray and green, respectively. And the longer the arrow length, the greater the relative hydrophobic moments in figure. Meanwhile, TSW-1 and TSW-2 have the best arrangement with a completely integrated hydrophobic face and cationic face. Furthermore, the helical wheel projections of others were added in supporting information (Figures S3).



Figure 2. Cytotoxicity of the engineered peptides against RAW 264.7 cells (A). The graphs were derived from an average of three independent trials. (B) Time-kill kinetic curves of the W8(B-1) and melittin (B-2) at 1 × MBC/MFC against E. coli ATCC 25922, S. aureus 29213 and C. albicans 2.2086. The graphs of kinetic were the mean values of three independent experiments. (C) Hemolytic activity of the engineered peptides and melittin against hRBCs. The graph was derived from the average of three independent trials. (D) Resistance proceedings in the presence of sub-MBC concentration of the peptides against P. aeruginosa ATCC 27853. The graph was from three independent experiments and each independent experiment contained three technical replicates.


Page 90 of 97

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Figure 3. (A) Clinical slit lamp scores against keratitis after twelve female mice per group were treated with four topical eye drop solutions: 0.7% saline solution (control), amphotericin B (1 mg/mL), W8 (5 mg/mL and 1 mg/mL). Moreover, the reference standard of clinical slit lamp scores as shown in Supplementary Table S1 and all of data are expressed as the mean ± s.d. *P

Rational design of short peptide variants by using ... - ACS Publications

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