Novel Design of Heptad Amphiphiles To Enhance Cell Selectivity, Salt

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Novel design of heptad amphiphiles to enhance cell selectivity, salt resistance, anti-biofilm properties and their membrane-disruptive mechanism Xiujing Dou, Xin Zhu, Jiajun Wang, Na Dong, and Anshan Shan J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01457 • Publication Date (Web): 23 Feb 2017 Downloaded from http://pubs.acs.org on February 23, 2017

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Journal of Medicinal Chemistry

Novel design of heptad amphiphiles to enhance cell selectivity, salt resistance, anti-biofilm properties and their membrane-disruptive mechanism Xiujing Dou1, Xin Zhu1,2, Jiajun Wang1, Na Dong1, and Anshan Shan1* 1

Institute of Animal Nutrition, Northeast Agricultural University, Harbin 150030, P. R. China

2

College of Animal Science & Veterinary Medicine, Shenyang Agricultural University, Shenyang, 110866, P. R.

China

ABSTRACT: Coiled-coil, a basic folding pattern of native proteins, was previously demonstrated to be associated with the specific spatial recognition, association, and dissociation of proteins and can be used to perfect engineering peptide model. Thus, in this study, a series of amphiphiles composed of heptads repeats with coiled-coil structures was constructed, and the designed peptides exhibited a broad spectrum of antimicrobial activities. Circular dichroism and biological assays showed that the heptad repeats and length of the linker between the heptads largely influenced the amphiphile’s helical propensity and cell selectivity. The engineered amphiphiles were also found to efficiently reduce sessile P. aeruginosa biofilm biomass, neutralize endotoxins, inhibit the inflammatory response and remain active under physiological salt concentrations. In summary, these findings are helpful for short AMP design with a highly therapeutic index to treat bacteria-induced infection. KEYWORDS: coiled-coil, antimicrobial peptide, cell selectivity, antibiotic resistance, P. aeruginosa

1

ACS Paragon Plus Environment


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INTRODUCTION The rise and spread of antibiotic-resistant bacteria, which are developing resistance to traditional treatments, are becoming a serious threat to public health seriously 1. The novel drug discovery and development rates are slowing, particularly in the field of antibiotics. Therefore, it is imperative to identify and design alternative novel antimicrobial agents that can eradicate resistant bacteria infection effectively. Antimicrobial peptides (AMPs) distributed in microorganisms, plants and animals are widely a fundamental element in the innate defense system 2. They have attracted great attention because they not only possess direct activity against bacteria, fungi, viruses, protozoa, and metazoan but also have indirect immune modulating activity in the host. Unlike conventional antibiotics, which function primarily by interacting with intracellular specific targets, the target of AMPs is usually the cell membrane by mechanistic and physical disruptions. However, developing modificated AMPs to reduce resistance is very difficult, because membrane disruption and/or independent on biochemical pathways would be required. Thus, using AMPs as a potent means of developing alternative traditional antimicrobial drugs is a potential avenue for drug discovery However, significant bottlenecks constraining the clinical application of AMPs exist, such as high manufacturing cost, poor stability and high systemic toxicity. To overcome these barriers, naturally occurring AMPs need to be optimized or modified using methods such as sequence truncation, substitution, or cyclization to generate new peptide analogs with enhanced antimicrobial activity and cell selectivity.

2

Unfortunately, the methods used for sequence optimization are largely empirical, making it even harder to decipher common structure-function relationships. More importantly, if the sequences of AMPs are too similar to naturally occurring AMPs,

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their application may trigger immunogenicity and ultimately compromise the host defense system, causing serious harm to the environment and public health 3. Therefore, the de novo design of peptides with minimal similarity to naturally occurring peptides is an effective way to exploit the safety and effectiveness of AMPs for clinical application. The heptad repeat sequence, is the reiteration of seven amino acids, indicated as “abcdefg”, in which hydrophobic residues are found at the “a” and “d” positions, and polar/charged residues are found at the other positions. This sequence motif often constitutes the main structure of coiled-coil peptides and has been widely identified in a number of natural proteins, such as DNA-binding proteins, cell surface proteins, transcription factors, tumor suppressors, among others. All of these molecules play important roles in specific functions as an optimal model for developing peptide biomaterials 4, 5. However, until now, little research has focused on utilizing this sequence motif to design AMPs. Herein, we designed a series of short heptad repeat sequence-based amphiphiles by maintaining the structural properties that are beneficial for antibacterial activity and cell selectivity. The sequence of the designed amphiphiles was (XRRXRRR)n-(H)n-(RRRXRRX)n, where X is Leu, Val, Phe, or Trp; R is Arg; H is linker (Trp); and n is number of repeat units (1, 2, or 3). The antimicrobial activities of the engineered amphiphiles against clinically relevant microorganisms, especially clinically isolated, drug resistant strains of P. aeruginosa, and the salt resistance, endotoxin neutralization and eradication of pre-formed biofilms were evaluated in relation to their cytotoxicities. Additionally, the mechanism of actions was examined using flow cytometry, scanning electronic microscopy assays (SEM) and transmission electronic microscopy assays (TEM), and so on. RESULTS AND DISCUSSION

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Design and characterization of the designed amphiphiles. Amidation modifications at the C-terminus were performed in all of the designed amphiphiles to offer a high net positive charge for enhanced antimicrobial activity. As shown in Table 1 and Figure S1, the molecular weights were determined using mass spectroscopy (MS) and the results were in close agreement with the anticipated results showing that the amphiphiles were successfully synthesized. Their cationic nature, amphipathicity and hydrophobicity have major impacts on the AMP antibacterial activity and cytotoxicity. For an amphipathic AMP, its positively charged patches participate in the original electrostatic interaction between AMPs and the bacterial cytomembrane, affecting cell selectivity, while the peptide penetrates into the lipid bilayer because of its hydrophobic patches, thereby permeating the membrane 6. In this study, the wheel diagram showed that Arg residues were segregated to the polar area, whereas some hydrophobic amino acids were segregated to the opposite area of the amphiphiles, yielding an amphipathic model (Figure 1), thus contributing to membrane attachment and disintegration. Many studies have demonstrated that the structural changes of AMPs under a variety of environments are associated with their biological activities

7-9

. Our results

indicated that under aqueous environments (e.g., PBS), each amphiphile had a random coil structure, showing a negative band at approximately 200 nm, except for 9 (LW3), which exhibited a second negative band at approximately 222 nm, indicative of a well-defined structure. In the presence of a 30 mM SDS micelle solution mimicking microbial membrane hydrophobic environments 10, all of the amphiphiles assembled into an α-helical structure (Figure 2 and Table S1). Notably, unlike other amphiphiles, 9 adopted a predominantly α-helical conformation in both PBS and SDS. A comparison of the molecular mean residual ellipticity values of the amphiphiles at a

4


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222 nm wavelength, which is an indicator of the α-helix content of a peptide, [θ]222 for 3 of -20,450 in PBS compared with -27,413 in SDS, while [θ]222 for melittin of -6,198 in PBS compared with -30,121 in SDS. These observations indicated that the α-helical structural rigidity of the peptide may play a crucial part in its antimicrobial and hemolytic activities. In addition, we found that peptides containing Leu, Ala, Phe, or Trp residues in heptads can form α-helical structures. However, [θ]222 value of FW (-20,334) was slightly less than that of 1 (LW) (-25,393), 2 (AW) (-23,867) and 4 (WW) (-25,873) in SDS, suggesting that Phe residues have weaker propensities for α-helical formation, This was also demonstrated by previous studies that Leu and Ala residues are strongly biased towards forming α-helical structures, while Phe is rather indifferent 3. Here, we also probed the effects of sequence symmetry on the antibacterial and cytotoxicity of the peptides. The order of the amino acid in heptads was rearranged to engineer non-mirror symmetric peptides that had the same amino acid composition, molecular weight, charge and hydrophobicity as its mirror symmetric counterparts. The CD results showed that 5 (LrW) with a non-mirror symmetric sequence manner exhibited lower α-helical content in SDS than 1 with a mirror symmetric sequence ([θ]222 for 5 of -15,023 compared with 1 of 25,393), suggesting that the symmetry of the sequence can affect the protein’s sability to from a helical structure for the amphiphile. Antibacterial and hemolytic activities of the designed amphiphiles. The antibacterial ability of the designed amphiphiles was detected against S. aureus and E. coli, as well as P. aeruginosa, including clinical and drug-resistant strains. As shown in Table 2, the designed amphiphiles displayed strong activities against S. aureus and E. coli. For P. aeruginosa strains, the geometric mean (GM) MICs ranged from 4.2 to

5



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194.0 mg/L. First, as seen in the results, among the amphiphiles incorporating Leu, Ala, Phe and Trp, Leu-containing 1 exhibited the most effective antimicrobial activities against the P. aeruginosa strains tested. The reasons for this were possibly the genetic or evolutionary preference for Leu at the “a” and “d” positions in the heptads and the importance of maintaining a certain magnitude of hydrophobicity/amphipathicity for sufficient antimicrobial activity. Net charge, hydrophobicity, amphipathicity and α-helical propensity are associated with the antibacterial activity of the α-helical AMPs 6. It is not simply the positive correlation of the net charge for the antimicrobial activity and there is a threshold. Previous studies have shown that net charges for +6 ― +7 for the peptide can be optimal for the antibacterial activity 7. The net charges of the designed amphiphiles were all higher than +6, indicating the sufficient driving force of the amphiphiles to bind to bacterial membranes, which are negatively charged. Furthermore, the relationship between hydrophobicity and biological activity has an analogous threshold. Increasing or decreasing the hydrophobicity above or below the threshold results in compromised antimicrobial activity. Compared to Leu, Ala, which has a small hydrophobic side chain, decreased peptide hydrophobicity/amphipathicity (Table 1), thus leading to weak membrane perturbation or permeabilization. Although Phe is highly hydrophobic, it has a lower affinity to form α-helicales. It was previously suggested that under membrane-mimicking environments, the bias to form an amphipathic α-helix is crucial for the membrane lytic activity of α-helical AMPs 6, and this may be the reason why 3 (FW) has a compromised antimicrobial activity. Trp has an indole side chain and can interact with the amino choline head group of the lipid bilayer, thus leading to a higher affinity for membranes and further insertion into the lipid bilayer 11. Therefore, Trp is frequently found in natural AMPs and is often

6


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used to improve their antimicrobial activities. However, in this study, we found that there was not an increase for the antibacterial activity of the peptide 4 after the introduction of Trp residues in heptads. Presumably at the “a” and “d” positions location of Trp residues in the heptad may partially hamper the packaging complementarity and burying hydrogen-bonding requirement, affecting the stability of the peptide structure and interaction between peptide and the membrane

12

, thus

leading to compromised antimicrobial activity. We also found that 5, which had a non-mirror symmetrical sequence pattern, had a lower activity against P. aeruginosa than 1, which had a mirror symmetrical sequence pattern, corresponding to its lower α-helical content in microbial membrane-mimetic environments (SDS) (Figure 2B). These data clearly demonstrated that the ability of the designed AMPs to form α-helical structures was crucial for conferring AMPs with potent antimicrobial activities, consistent with previous reports that found that the antimicrobial activity of AMPs benefited from disconnecting the hydrophobic clusters and polar residues upon secondary structures formation, leading to the disruption of the membrane lipid bilayers 8. Next, to investigate the effects of the sequence length on the antimicrobial properties, the heptad of 1 was increased to n = 2 (6 (L2W)) and n = 3 (7 (L3W)). As shown in Table 2, 6 and 7 displayed markedly improved antimicrobial activities, regardless of the strains tested. According to the CD data (Figure 2) and previous work, α-coil structure formation in anionic hydrophobic environments (SDS), which is implicated in the oligomerization and/or the self-assembly of a peptide or protein, may give 7 with a greater degree of bacterial membrane interaction and perturbation, which is essential for the stabilization of membrane pores and ion channels, thus leading to enhanced antimicrobial activity 5, 13. Additionally, we further determined the

7



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influence of the linker length (Trp residues) on the antipseudomonal activities of the amphiphiles. As shown in Table 2, the antipseudomonal activity of 2, which had a linker length of n = 2, was increased compared to that of 1, while that of 9, which had a linker length of n = 3, was the highest among all of our designed amphiphiles. In combination with the CD results, the structural rigidity of 9), which helped it form an α-helix in both aqueous and membrane-mimicking environments, as shown in Figure 2, may be beneficial to its antibacterial activity. Previous reports have suggested that linear cationic α-helical peptides with enhanced structural rigidity could more easily form a membrane pore and penetrate the membrane

14

. In addition, the increased

hydrophobicity, disruptive amphipathicity and location of Trp residues in the middle position of the peptide sequences may also contribute to enhanced antimicrobial potency and cell selectivity, as reported in previous studies 11, 15, 16. The hemolytic activities of the designed amphiphiles were evaluated using hRBCs. Compared with melittin, minor differences in the hemolytic activities between these synthetic amphiphiles were found (Figure 3). Although 6 and 7 showed a slight concentration-dependent toxicity against hRBCs, the concentrations required for the induction of hemolysis were far above their MIC values (Table 2). We further determined as the lowest peptide concentration that induced hemolysis) to the GM of the MICs. Larger TI values corresponded to greater cell selectivity. All the designed amphiphiles displayed much higher TI values than melittin, as shown in Table 2. In particular, among the designed amphiphiles 9, because of its relatively higher antimicrobial activity and lower hemolytic ability, showed the highest TI value. It was followed by 8 (LW2), which had significantly greater TI values than the clinically -available antipseudomonal drugs ciprofloxacin, gentamicin and ceftazidime, demonstrating that 8 and 9 are strong therapeutic candidates against drug-resistant

8


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microbial infections. Antimicrobial mechanism. The amphiphiles containing Leu residues were further studied for their antimicrobial mechanism on the basis of their MIC and MHC values. As shown in Figure 4, the designed amphiphiles effectively enhanced the outer membrane permeability of P. aeruginosa in a concentration-dependently manner, indicating the strong ability of the AMP to overcome the barrier determine the susceptibility of the microorganism to compound. The cytomembrane is always considered to be the primary target of AMPs. Upon perturbation and permeabilization of microbial membranes, the electrical potential of the cytoplasmic membrane will dissipate because of pore formation and ion channel generation 18. As shown in Figure 5, the optimized sequences for 7 and 9 caused cytoplasmic membrane of P. aeruginosa to depolarize rapidly in a concentration-dependent manner. Flow cytometry analysis further demonstrated that, compared to melittin, these amphiphiles damaged the cell membrane integrity at their 1 × MICs (Figure 6). Notably, compared to 1, 5 showed a weaker ability to induce cytoplasmic membrane depolarization and damage the integrity of the P. aeruginosa membrane (Figure 5 and Figure 6), suggesting that the propensity and/or extent to form an α-helical structure is crucial to the membrane-lytic activity of AMPs under membrane-mimicking environments (Figure 2B). The surface morphologies and intracellular changes in bacterial cells after treatment with amphiphiles were investigated using SEM and TEM. In Figure 7A, we observed obvious membrane distortion, corrugation, and damage on the surface of P. aeruginosa cells after a short 1-h treatment with amphiphiles at 1 × MICs compared to the smooth surface of the control. In addition, we also observed bacterial membrane damage and the outflow of intracellular contents after treatment with 9 (Figure 7B). Furthermore, the cellular localization of the peptides was further

9



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investigated using fluorescently labeled peptides with confocal laser-scanning microscopy in P. aeruginosa cells. As seen in Figure 8, fluorescently labeled peptide 8 and 9 accumulated at the cell surface which was similar to melittin, a peptide located on membranes. This result further revealed that 8 and 9 were capable of binding to the membrane of P. aeruginosa effectively. These observations indicated that the optimized amphiphiles can readily permeabilize the outer membrane of the bacterial membrane after perturbing the membrane structure, thus resulting in the death of the bacterial. Conventional antibiotics inhibit key intracellular targets involved in biosynthetic pathways in microorganisms, while AMPs disrupt the bacterial cell membranes rapidly through mechanical measures; it is thought this is what helps overcome antibiotic resistance resulting from conventional antibiotics 11, 13. Endotoxin neutralization activities. LPS, also called endotoxin, is an important cell wall component of Gram-negative bacteria that causes a systemic inflammatory response that accompanies bacterial infections 17. Many studies have demonstrated that AMPs have the ability to firmly bind LPS, disrupt LPS aggregates, and lower LPS-induced pro-inflammatory responses, making them particularly useful candidates for anti-sepsis drugs

17, 19-21

. Therefore, we further investigated the abilities of the

optimized amphiphiles to bind LPS using a BODIPY-TR-cadaverine displacement assay. The fluorescence of BODIPY-TR-cadaverine is quenched when it binds to LPS, and the removal of BODIPY-TR-cadaverine by the amphiphiles results in fluorescence. As seen in Figure 9A, 8 and 9 showed a concentration-dependent increase in the fluorescent intensities, clearly demonstrating the abilities of the amphiphiles to bind LPS. SPR spectroscopy was used to further investigate the real-time and direct binding interaction between the peptides and LPS. As shown in Figure 9B, 9C, and 9D, the sensorgram for the binding of different levels of LPS and

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peptides illustrated that the response unit of LPS binding to immobilized 8, 9, and melittin was enhanced rapidly. Moreover, the binding of LPS to 8, 9, and melittin had an obvious dose-dependent pattern at concentrations 3.125, 6.25, or 12.5 µg/mL. Herein, 8 and 9 were shown to strongly interact with LPS via SPR data 22. The endotoxin-neutralizing capability of the optimized amphiphile sequences was further evaluated in RAW264.7 macrophages provoked with LPS in the presence of amphiphiles. Nitric oxide (NO), a representative product of the inflammatory response that occurs after stimulation with LPS, is secreted by monocytes and macrophages 17. The nitrite levels secreted by macrophages treated with LPS in presence of the amphiphiles were detected to confirm the LPS neutralization ability compared to the levels without the peptides. As shown in Figure 10A, the optimized sequences of 8 and 9 effectively inhibited NO production at 10 mg/L. In addition, the LPS-activated inflammatory response is always accompanied with by inflammatory cytokines production, including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), among others 23. The ELISA and real-time PCR results showed that 8 and 9 effectively attenuated the protein level of TNF-α and mRNA expression of TNF-α, IL-6, and IL-1β in the cytoplasm in LPS-stimulated macrophages (LPS at 100 ng/mL) (Figure 10B, 10C, 10D and 10E). LPS triggers downstream signaling activation by interacting with the targeted receptors. Among the receptors, toll like receptor 4 (TLR4) is the main receptor. Inflammatory factor production is often increased through TLR4 recognition and the activation of the transcriptional factor NF-κB pathway 24, 25. In this study, Western blots were performed to further study the changes in the important signaling proteins. The results demonstrated that 8 and 9 effectively relieved the response induced by LPS from P. aeruginosa, in RAW264.7 cells. As depicted in figure 9F, after 18 h of treatment, LPS

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alone induced TLR4 negative regulation and the degradation of IκBα protein and increased the expression of inducible nitric oxide synthase (iNOS). However, these series of action were weakened or restored effectively by treatment with 8 and 9, suggesting that the negative regulation of TLR4 was blocked by 8 and 9 in LPS-induced RAW264.7 macrophages. Additionally, the IκBα pathway and production of iNOS were inhibited. Furthermore, the production of NO and inflammatory cytokines was inhibited effectively. Importantly, we also found that the synthetic amphiphiles did not significantly reduce cell viability when used at 10 mg/L (Table 2). These results confirmed that the optimized amphiphiles possessed well-defined anti-inflammatory properties and inhibited the production of NO and pro-inflammatory factors via the direct scavenging and modulation of cellular inflammatory pathways, indicating their potential for systemic administration to treat bloodstream infections. Salt resistant activities. Maintaining the activity of AMPs in physiological environments is required in a therapeutic setting. However, antagonism between peptides and ionic strength occurs frequently, which seriously hinders the application of AMPs as novel antimicrobial drugs 26. As shown in Table 3, under physiological concentrations of cations (Na+, K+, NH4+, Zn2+, Fe3+), the antimicrobial activities of 8 and 9 were not markedly affected. However, the amphiphiles exhibited a slightly compromised activity when divalent Mg2+ was present. Many studies have previously demonstrated that monovalent cations, such as Na+ and K+, can inhibit the antimicrobial activities of AMPs to various extents

27, 28

. Usually, peptides that are

positively charged and membranes that are negatively charge are attracted each other, which is directly inhibited by monovalent cations. However, for divalent or multivalent cations, they stabilize the rigid outer membrane by binding to anionic

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phosphate groups, such as those in LPS or lipoteichoic acid (LTA), thus hampering membrane binding and the uptake of more peptide molecules and presenting more antagonistic activity than monovalent cations 29. However, this antagonistic effect is not always the case. Some studies reported that AMPs with a low net positive charge were more affected by divalent cations than high net positive charges AMPs Moreover,

divalent

cations

at

a

low

concentration

could

improve

30

.

the

peptide-membrane interaction. On the contrary, the excessive electrostatic interactions caused by the high concentration of divalent cations increased the membrane rigidity, slowly hindering pore formation 31. Therefore, it can be concluded that divalent or multivalent cations have a counter effect in a peptide- or concentration-dependent manner. Antibiofilm activities. Bacteria growing on a surface can often form biofilms, causing a variety of infections. The presence of biofilms may provide protection from eradication by the host defense system or antimicrobial agents, thus hindering treatment

32, 33

. Indeed, biofilm cells possess a stronger resistance to the majority of

antibacterial drugs compared to planktonic cells 34, 35. Thus, we further determined the antibiofilm abilities of the designed amphiphiles using a biofilm model. As shown in Figure 11A, the biomasses of biofilms treated with 8 and 9 were decreased in a concentration-dependent manner. SEM further indicated that biofilms exhibited observable interconnected matrices or conditioning layers in the absence of amphiphiles. By contrast, the cell densities of the biofilms were significantly reduced after treatment with amphiphiles at 4 × MICs (Figure 11B). Taken together, these results thus provide direct evidence that the designed amphiphiles have the ability to work against bacterial biofilms and induced the outflow of biofilms efficiently. CONCLUSION

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In this study, a series of α-helical amphiphile amphiphiles containing coiled-coil heptads was synthesized and analyzed for their antimicrobial, anti-biofilm and anti-endotoxin properties. Among the synthetic amphiphiles, amphiphile (1) with Leu at the “a” and “d” positions in the heptads were significantly more potent than amphiphiles with Ala (2), Phe (3), and Trp (4) at these positions. Amphiphile sequences with n = 2 and n = 3 repeating units of the heptad, named L2W and L3W, respectively, were shown to possess high anti-pseudomonal activity. However, they also exhibited slight cytotoxicity and were hemolytic at concentrations far above their MICs. Furthermore, an increased linker length, such as those in 8 and 9, conferred increased activity and selectivity to the amphiphiles. The optimal 9 also demonstrated efficient endotoxin binding, salt resistance, biofilm dispersion, and membrane depolarization and disrupting capabilities, with no or minimal cytotoxicity. In conclusion, this study clearly proved that by utilizing a natural sequence model, we rationally engineered amphiphiles with heptads from coiled-coil that are highly selective. These amphiphiles are powerful candidates for bacterial infection-related applications, such as peptide-based biomaterial engineering, biomedical coatings, antimicrobial agents and immune-regulatory drugs. EXPERIMENTAL SECTION Materials. The peptides used in this study were synthesized by GL Biochem (Shanghai, China) and purified to more than 95% using reverse-phase high performance liquid chromatography (RP-HPLC) (Supporting Information, Figure S1). The molecular weights of the peptides were further confirmed using mass spectroscopy (MS). Peptides were dissolved in pure water at a concentration of 2.56 mg/mL and the peptide solutions were stored at -20 °C before subsequent assessments.

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E. coli ATCC25922, S. aureus ATCC29213, and P. aeruginosa ATCC27853 were kept in our laboratory, P. aeruginosa CICC10419, CICC21625 and CICC21630 were kindly provided by Professor Jianhua Wang (Chinese Academy of Agriculture Sciences, Beijing, China). Ciprofloxacin- (11411), gentamicin- (11421), and ceftazidime- (11431) resistant variants of P. aeruginosa were obtained from the College of Veterinary Medicine, Northeast Agricultural University (Harbin, China). Clinical isolates of P. aeruginosa 21328, 25349 and 26305 were provided by the 2nd Affiliated Hospital of Harbin Medical University (Harbin, China). All the stains were stored at -80°C in 15% glycerol, cultured at 37°C on Mueller-Hinton (MH) agar, and grown to the exponential phase in MH broth with rotary shaking at 220 rpm before use. The murine macrophage cell line RAW264.7 was purchased from the cell bank of the Chinese Academy of Sciences, SIBS (Shanghai, China). Phosphate-buffered saline (PBS), sodium dodecyl sulfate (SDS), and trifluoroethyl alcohol (TFE) were purchased from Solarbio (China), Sigma-Aldrich (China), and Amresco (USA), respectively. MH broth (MHB) powder was purchased from AoBoX (China) and used to prepare the culture medium according to the manufacturer’s instructions. Sodium chloride, potassium chloride, ammonium chloride, zinc chloride, magnesium chloride, ferric chloride, and glucose were purchased from Kermel (analytical grade, China). Triton X-100, EDTA, HEPES, N-phenyl-1-napthylamine (NPN), 3.3’-depropylthiadicarbocyanine iodide (diSC3-5), propidium iodide (PI), lipopolysaccharide (LPS, derived from P. aeruginosa 10), polymyxin B, 3-(4,5-demethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. Ethanol, acetone, tertiary butanol, and glutaraldehyde were all analytical grade (Sigma-Aldrich). The Griess reagent system was purchased from Promega (USA). RPMI 1640 and fetal bovine

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serum (FBS) were purchased from Invitrogen (USA). A mouse TNF-α ELISA kit was purchased from Boster (EK0527, China). The antibodies against IκBα (SC-371, Santa Cruz) and β-actin (13E5) and the secondary horseradish peroxidase (HRP)-conjugated anti-rabbit IgG were purchased from Cell Signaling Technology, Inc. (4970, 7077, USA); Antibodies against TLR4 and iNOS were purchased from Abcam (ab8376, ab178945, respectively; UK). Peptide design and sequence analysis. We adopted a facile approach to design a series

of

short

heptad

amphiphiles

consisting

of

short

recurring

(XRRXRRR)n-(H)n-(RRRXRRX)n sequences. As implied from the natural heptad sequence model “abcdefg”, of which hydrophobic residues are in “a” and “d” positions, and hydrophilic residues are in other positions

4, 5

, we placed hydrophobic

residues in “X” positions and hydrophilic residues in “R” positions in a heptad. Additionally, previous studies have shown that a centrosymmetric distribution of amino acids might be a relic of evolutionary divergence and contributed to the antimicrobial activity of a peptide7, 36-38. Thus, we put a linker in the middle position of the peptide, linked with heptad repeat sequences in a mirror-like symmetric manner, with the aim to facilitate a conformational flexibility and increase a membrane interaction. In addition, another several basic design principles were also included: 1) Different types of hydrophobic residues (Leu, Ala, Phe and Trp) with varied polarity and bulkiness of the hydrophobic side chain were placed at “X” positions of a heptad. Leu is an aliphatic amino acid and has a high hydrophobicity, Ala is low hydrophobic but has a high helicity. Phe is an aromatic amino acid. Trp is a heterocyclic amino acid. They represent different structural characteristics of amino acids and were chosen to look into the influence on the antimicrobial and cytotoxic activities of these peptides.

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2) The incorporation of cationic Arg residues in “R” positions of a heptad was intended to provide adequate driving force to the negatively charged compounds of the bacterial membrane and improved antimicrobial activity than Lys residue

39, 40

. 3)

The introduction and location of Trp residue, as the linker, in the middle position of paired heptads was to enable the peptide’s membrane-seeking ability and facilitate to form defined structures 15, 16, 41. 4) The method of repeat stacking has been shown to be an effective way to optimize and/or design AMPs 7, 37. Thus, increasing the number of repeat units was investigated for effects of length of peptide sequence on biological activities of the designed amphiphiles. The primary sequence analysis of the peptides was performed online using EMBOSS Pepinfo

9, 42

online using Jpred 4

. The secondary structure type of the peptides was predicted

43, 44

. The charge, hydrophobicity and hydrophobic moment were

calculated online using HeliQuest

9, 45

. The helical wheel projection was performed

online using the Helical Wheel Projections

9, 46

. The percentages of α-helix, β-sheet,

turn, and arbitrary coil of samples in PBS and SDS solutions were calculated using K2D3 47. Circular dichroism (CD) spectroscopy. Each peptide was dissolved in 10 mM PBS or 30 mM SDS to give a final concentration of 150 µM, as described previously 11, 48

. All CD spectra were obtained by three scans on a J-820 spectropolarimeter (Jasco,

Tokyo, Japan) using a rectangular quartz cell with a path length of 0.1 cm from 190 to 250 nm at 10 nm/min at room temperature. The acquired spectra were then converted to the mean residue ellipticity by using the following equation: θM= (θobs• 1000) / (c • l • n) where θM is the mean residue ellipticity (deg • cm2 • dmol-1), θobs is the observed ellipticity corrected for the buffer at a given wavelength (mdeg), c is peptide

17



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concentration (mM), l is the path length (mm) and n is the number of amino acids. Antimicrobial assay. The minimal inhibitory concentration (MIC) assay was performed using the broth microdilution method16. Briefly, bacteria were grown to the exponential growth phase in MHB at 37 °C, and diluted to approximately 1 × 106 CFU/mL in the same broth. 50 µL of bacterial inoculum was added in 96-well microtiter plates with 50 µL of two-fold diluted peptides. MICs were defined as the lowest concentration that inhibited visible turbidity by visual inspection after incubation at 37°C for 18 h. Uninoculated MHB was used as the negative control, and cultures without added peptides served as the positive control. Each test was reproduced at least three times using two replicates each. This assay was also performed in the presence of monovalent cations (Na+, K+, and NH4+), divalent cations (Mg2+ and Zn2+), or a trivalent cation (Fe3+), in the form of chloride salts, at physiological concentrations to investigate the effects of cations on the antimicrobial activity of synthetic peptides. Hemolytic assay. Fresh human red bold cells (hRBCs) were collected and diluted 10-fold in PBS (pH 7.4). Then 100 µL of hRBCs was added to each tube containing an equal volume (100 µL) of peptide in PBS. After incubation at 37 °C for 1 h, the suspension was centrifuged at 1 000 × g for 5 min, and the supernatants were transferred to wells of a 96-well plate. Release of hemoglobin was monitored by measuring the absorbance at 570 nm. The hRBCs in PBS and 0.1% Triton X-100 were employed as negative and positive controls, respectively. The percent hemolysis was calculated using the following formula: Percent hemolysis = [(A-A0)/(At-A0)] ×100 where A represents the absorbance of the peptide sample at 576 nm, A0 and At represented the absorbance at 0% and 100% hemolysis as determined in 10 mM PBS

18


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and 0.1% Triton X-100, respectively. Each test was reproduced at least three times using two replicates. The method was designed according to a previously described method 16. Membrane permeability assay. The fluorescent dye NPN assay was used to determine the outer membrane permeability as previously described 49. Briefly, P. aeruginosa ATCC27853 cells at the exponential phase were washed three times with HEPES buffer (pH 7.4) containing 5 mM glucose and diluted to approximately 105 CFU/mL in the same buffer. A final concentration of 10 µM NPN was added to the bacterial suspension, and the background fluorescence was recorded using an F-4500 fluorescence spectrophotometer (Hitachi, Japan) at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. A different final concentration of the peptides was added to the cuvette, and the fluorescence was monitored until no further increase in fluorescence was observed. Polymyxin B (10 mg/L) was used as a positive control (F100) because of its strong outer membrane permeabilization. Each test was reproduced at least three times using two replicates. The values were converted to % NPN uptake using the equation: % NPN uptake = (Fobs - F0)/(F100 - F0) ×100 where Fobs is the observed fluorescence at a given peptide concentration, F0 is the initial fluorescence of NPN in the absence of the peptide and F100 is the fluorescence of NPN upon the addition of 10 µg/ml of polymyxin B (Sigma). “100%” represents “% NPN uptake” of the positive control, polymyxin B. The cytoplasmic membrane electrical potential variation upon peptide treatment was measured using the membrane potential-sensitive dye diSC3-5 as described before 18. Briefly, P. aeruginosa ATCC27853 cells grown to the exponential phase were collected, washed three times with 5 mM HEPES buffer (pH 7.4), and

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resuspended in the same buffer containing 0.3 mM EDTA to an OD600 of 0.05. diSC3-5 and KCl were added to the cell suspension at final concentrations of 0.4 µM and 0.1 M, respectively, and samples were then incubated at 37 °C for 90 min in the dark. Different concentrations of the peptides were added and the fluorescence was monitored at an excitation wavelength of 622 nm and an emission wavelength of 670 nm. Each test was reproduced at least three times using two replicates. Membrane integrity assay. Membrane integrity of the peptides against P. aeruginosa was determined as previously described 9. P. aeruginosa ATCC27853 cells were grown to mid-logarithmic phase in MHB, washed three times with PBS (pH 7.4), and resuspended in the same buffer to obtain a 105 CFU/mL bacterial suspension. A final concentration of 1 × MIC of peptides was added to the suspension, and the mixtures were incubated at 37°C for 15 min. Samples were then fixed with PI (final concentration 25 µg/mL) at room temperature for 15 min in the dark, followed by removal of the unbound dye through washing with an excess of PBS. Cells stained by PI were examined using fluorescence-assisted cell sorting (FACS) with a flow cytometer (Becton-Dickinson, USA) at a laser excitation wavelength of 488 nm. Membrane integrity observations. Scanning electronic microscopy (SEM) was used to evaluate the surface morphology alterations of bacterial cells after peptide treatment. P. aeruginosa ATCC27853 cells in the exponential phase were harvested, washed twice with PBS (pH 7.4), and resuspended to reach an OD600 of 0.2. Cells were treated with peptides at their 1 × MICs for 60 min at 37°C. After incubation, the cell pellets were harvested, washed with PBS twice and subjected to fixation with 2.5% glutaraldehyde at 4 °C overnight. The cells were dehydrated for 10 min in a graded ethanol series (50%, 70%, 90%, and 100%) followed by 15 min in 100% ethanol, a mixture (1:1) of 100% ethanol and tertiary butanol and absolute tertiary butanol.

20


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Finally, the specimens were dehydrated, dried, coated with gold, and examined using a Hitachi S-4800 SEM (Japan). Each test was reproduced at least three times using two replicates. Transmission electronic microscopy (TEM) was used to evaluate the morphological and intracellular alterations of bacterial cells after peptide treatment. Samples were prepared for TEM in the same manner as for SEM. After pre-fixation with 2.5% glutaraldehyde at 4°C overnight, the cells were washed with PBS (pH 7.4) twice and post-fixed with 2% osmium tetroxide for 70 min. After dehydration with a graded ethanol series (50%, 70%, 90%, and 100%) for 8 min each, the bacterial samples were transferred to a mixture (1:1) of 100% ethanol and acetone, followed by absolute acetone for 10 min. The specimens were then transferred to a mixture (1:1) of absolute acetone and epoxy resin for 30 min and then to a final epoxy resin overnight. Finally, the specimens were sectioned with an ultramicrotome, stained by uranyl acetate and lead citrate, and observed using a HITACHI H-7650 TEM. Each test was reproduced at least three times using two replicates.

Confocal laser scanning microscopy. P. aeruginosa (ATCC 27853) cells were incubated with FITC-labeled peptides and observed using confocal laser scanning microscopy to further analyze the cellular distribution of the peptides as previously described 16. P. aeruginosa cells (OD600 = 0.2) were incubated with the FITC-labeled peptides at 1 × MIC at 37 ˚C. After incubation for 30 min, the cell pellets were collected by centrifugation at 5,000 g for 5 min and washed three times with PBS buffer. A smear was made, and images were captured using a Leica TCS SP2 confocal laser scanning microscope with a 488-nm band pass filter for FITC excitation.

Biofilm biomass assay and imaging. A static abiotic solid surface assay was used

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to analyze biofilm formation as described before

16

. Overnight cultures of P.

aeruginosa ATCC27853 diluted 1:100 in fresh medium, added (500 µL) to wells of 12-well cell culture plates, and grown for 24 h at 37°C. Planktonic cells and loosely attached cells were removed, and wells were then replenished with 500 µL of fresh broth in the absence or presence of different concentrations of peptides. After incubation for 24 h, the culture medium was aspirated and the biofilms were stained with 500 µl of 0.1% (w/v) crystal violet for 10 min at room temperature. The excess crystal violet dye was removed by rinsing the wells with PBS five times. The wells were dried for 20 min and then refilled with 500 µL of 95% (v/v) ethanol to extract the dye that was associated with the biofilm. After 10 min, a 100 µL aliquot from each well was transferred to a new 96-well plate for quantification of absorbance at a wavelength of 600 nm using a microplate reader. The percent formation of a biofilm was calculated using the following formula: Percent formation of a biofilm = [(A-A0)/(At-A0)] ×100 where A represented the absorbance of the peptide sample at 600 nm, A0 and At represent the 0% and 100% formation, respectively, of a biofilm determined in 95% (v/v) ethanol to extract the dye that was associated with the biofilm. The experiments were reproduced at least three times in duplicate, and the data were expressed as the mean ± standard deviations. For microscopic observation of the biofilms, sterilized coverslips were placed at the bottom of a 12-well plate. After incubation, cells were fixed with 2.5% glutaraldehyde overnight. The coverslips were rinsed three times with PBS, and the samples were dehydrated through a graded series of ethanol as described above, followed by critical-point drying, gold sputtering, and examination using an SEM (Hitachi, Japan) at least three times in duplicate. 22


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LPS neutralization assays. The synthetic peptides were examined for LPS neutralizing activity in a BODIPY-TR-cadaverine displacement assay 18. Briefly, P. aeruginosa LPS and BODIPY-TR-cadaverine at final concentrations of 25 µg/mL and 2.5 µg/mL, respectively, were added into 50 mM of Tris buffer (pH 7.4). Two milliliters of this mixture was added to a quartz cuvette, and the fluorescence was recorded at an excitation wavelength of 580 nm and an emission wavelength of 620 nm with an F-4500 fluorescence spectrophotometer (Hitachi, Japan). The experiment was reproduced at least three times using two replicates. Surface plasmon resonance (SPR) experiments. The real-time binding interaction between peptides and LPS was measured by SPR using a Biacore 3000 instrument (GE Healthcare) 22. Briefly, the peptides were covalently immobilized on certified grade CM5 sensor chips at 20 µg/mL in 10 mM sodium acetate buffer (pH 6.0), which is nearly 1300 resonance units (RUs) of the peptide, using the amine coupling kit according to the manufacturer's instructions. The unreacted moieties on the surface were blocked with ethanolamine. All measurements were carried out in 20 mM Tris (pH 7.4) and 100 mM NaCl. LPS in running buffer (20 mM Tris and 100 mM NaCl, pH 7.4) was flowed over the surface at 3.125 µg/mL-12.5 µg/mL with a flow rate of 10 µL/min. After each injection, the surface was regenerated with 50 mM NaOH containing 0.05% (w/v) SDS. Each test was reproduced independently three times. To investigate further the LPS neutralizing properties of the peptides, LPS-induced nitric oxide (NO) production was evaluated in the murine macrophage cell line RAW264.7 50. Briefly, RAW264.7 cells were plated at a density of 5 × 105 cells per well in 12-well plates and stimulated with LPS (100 ng/mL) in the absence or presence of peptides (10 mg/L) for 18 h at 37°C. Isolated supernatant fraction were

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mixed with an equal volume of Griess regent according to the manufacturer’s protocol, and samples were incubated at room temperature for 10 min. Nitrite production was measured by determining the absorbance at 540 nm, and the concentrations determined using a standard generated with NaNO2. Each test was reproduced independently at least three replicates. Cytotoxicity assays. The MTT assay was performed according to a previously described

. Briefly, RAW 264.7 cells (5 × 105/well) in RPMI 1640 medium

51

(supplemented with 10% FBS) were seeded into 96-well plates and then incubated in humidified atmosphere of 95% air and 5% CO2 at 37°C overnight. The next day, the peptides were added to the cell cultures at final concentrations of 0.25 - 256 mg/L. After incubation for 24 h, the cell cultures were incubated with MTT (50 µl, 0.5 mg/ml) for 4 h at 37 °C. The cell cultures were centrifuged at 1,000 × g for 5 min, and the supernatants were discarded. Subsequently, 150 µL of DMSO was added to dissolve the formed formazan crystals, and the absorbance was measured using a microplate reader (TECAN, Austria) at 570 nm. Each test was reproduced at least three times using two replicates. Cytokine levels assays. Quantitative real-time PCR (qRT-PCR) was performed to evaluate and quantify the mRNA levels of cytokines following the treatment described above in RAW264.7 cells 52. Briefly, the total RNA was isolated from RAW264.7 cells using TRIzol (Invitrogen) according to the manufacturer’s instructions. 1 µg total RNA was reverse transcribed using M-MLV reverse transcriptase (Takara, Japan). The cDNA samples were analyzed by qRT-PCR using SYBR Green I as the fluorescent dye (Takara, Japan). The relative quantifications of the mRNA expression of the target genes were calculated using the comparative threshold cycle number for each sample (2-∆∆CT). The gene expression was normalized to the corresponding mGAPDH level.

24


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The main primers used for qRT-PCR in this study are shown in Table S2. Each test was reproduced at least three times using three replicates. The ELISA assays to assess the level of TNF-α secreted by cultured RAW264.7 cells in the culture medium were performed using commercial kits. The experiments were performed in accordance with the manufacturer’s instruction, and each sample was analyzed in duplicate. The relative cytokine release was normalized to that of untreated controls. Signaling molecular assays. Western blots were performed to evaluate the receptor and signaling molecular protein levels using previously described methods 52. The cells were harvested and lysed with cold cell lysis buffer containing 1% protease inhibitor (Beyotime, USA). The total lysate was separated on an 8% or 12% SDS-PAGE gel and then transferred to PVDF membranes. The membranes were blocked with 5% non-fat dry milk in Tris-buffered saline (10 mM Tris-Cl at pH 7.5 and 150 mM NaCl) containing 0.05% Tween 20 (TBST) at 37 °C for 2 h and then incubated with the appropriate primary antibody at 4 °C overnight. Detection was performed using a horseradish peroxidase (HRP)-linked secondary antibody at room temperature for 1 h. Blots were developed using an enhanced chemiluminescence detection system (Applygen, China). The main antibodies used for Western blot in this study were including TLR4, IκB-α, iNOS and β-actin. Each test was reproduced for at least two replicates. Statistical analysis. The data were analyzed by ANOVA with the GLM procedure in the SAS 9.3 software (SAS Institute, Inc., Cary, NC, USA). Quantitative values were expressed as the mean ± standard errors. Differences were defined as significant at a P-value of less than 0.01. ANCILLARY INFORMATION Supporting Information.

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Figure S1: Details of the HPLC and MS characterization of the peptides. Figure S2: Helical wheel projection of the peptides. Table S1: Quantified estimation of the levels of helix, β-sheet, and random coil in the designed peptides. Table S2: List of primers used for qRT-PCR. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel: +86 451 55190685. Fax: +86 451 55103336. Address: 1 No. 59 Mucai Street Xiangfang District, Harbin, 150030, P. R. China Notes The authors declare no competing financial interest. Author Contributions X. J. Dou and X. Zhu contributed equally to this work, and they are both co-first authors. X. J. Dou and X. Zhu conceived and performed most of the experiments, including circular dichroism spectroscopy, the antimicrobial assay, the hemolytic assay, the membrane permeability assay, and so on. J. J. Wang and N. Dong performed part of the experiments, including the confocal laser scanning microscopy and SPR. A. S. Shan supervised the work. A. S. Shan and N. Dong revised the final version of the manuscript. All of the authors have read and approved the final version of the manuscript. ACKNOWLEDGEMENTS This work was funded by the National Natural Science Foundation of China (31472104, 31272453), the China Agriculture Research System (CARS-36), the 26


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Program of Ministry of Education of China (20092325110009) and the Program for Universities in Heilongjiang Province (1254CGZH22, 11551z003). We would be grateful to Yuanyuan Chen, Zhenwei Yang (Institute of Biophysics, Chinese Academy of Sciences) for technical help with Biacore experiments. ABBREVIATIONS USED AMPs, antimicrobial peptides; CD, circular dichroism; DMSO, dimethyl sulfoxide; EDTA, ethylenediaminetetraacetic acid; FACS, Fluorescence-assisted cell sorting; FBS, fetal bovine serum; hRBCs, human red bold cells; LPS, lipopolysaccharides MHB, Mueller−Hinton broth; MIC, minimum inhibitory concentration; MTT, 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetrazolium

bromide;

NF-κB,

nuclear

factor–κB; NO, nitric oxide; NPN, N-phenyl-1-napthylamine; OD, optical density; PI, propidium iodide; qRT-PCR, quantitative real-time PCR; SDS, sodium dodecyl sulfate; SEM, scanning electronic microscopy; TBST, tris-buffered saline; TFE, trifluoroethyl alcohol; TLR4, Toll-like receptor-4

27



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design of mirror-like peptides with alanine regulation. Amino Acids 2016, 48, 1-15. (39)

Veiga, A. S.; Sinthuvanich, C.; Gaspar, D.; Franquelim, H. G.; Castanho, M.

A.; Schneider, J. P. Arginine-rich self-assembling peptides as potent antibacterial gels. Biomaterials 2012, 33, 8907-8916. (40)

Torcato, I. M.; Huang, Y. H.; Franquelim, H. G.; Gaspar, D.; Craik, D. J.;

Castanho, M. A. R. B.; Henriques, S. T. Design and characterization of novel antimicrobial peptides, R-BP100 and RW-BP100, with activity against Gram-negative and Gram-positive bacteria. Biochim. Biophys. Acta. 2013, 1828, 944-955. (41)

Deslouches, B.; Phadke, S. M.; Lazarevic, V.; Cascio, M.; Islam, K.;

Montelaro, R. C.; Mietzner, T. A. De Novo Generation of Cationic Antimicrobial Peptides: Influence of Length and Tryptophan Substitution on Antimicrobial Activity. Antimicrob. Agents Chemother. 2005, 49, 316-322. (42)

EMBOSS Pepinfo homepage.

http://www.ebi.ac.uk/Tools/seqstats/emboss_pepinfo/ (accessed: March 12th 2016).

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(43)

Ma, Z.; Wei, D.; Yan, P.; Zhu, X.; Shan, A.; Bi, Z. Characterization of cell

selectivity, physiological stability and endotoxin neutralization capabilities of α-helix-based peptide amphiphiles. Biomaterials 2015, 52, 517-530. (44)

Secondary structure type of the peptides prediction online homepage.

http://www.compbio.dundee.ac. uk/jpred/index.html (accessed: March 12 2016). (45)

HeliQuest calculation homepage. http://heliquest.ipmc.cnrs.fr/ (accessed:

March 12 2016). (46)

Helical

wheel

projection

homepage.

http://rzlab.ucr.Edu

/scripts/wheel/wheel.cgi (accessed: March 12 2016). (47)

K2D3

webpage.

http://cbdm-01.zdv.uni-mainz.de/~andrade/k2d3//info/about.html

(accessed:

December 17 2016). (48)

Wiradharma, N.; Liu, S. Q.; Yang, Y. Y. Branched and 4-Arm Starlike α

-Helical Peptide Structures with Enhanced Antimicrobial Potency and Selectivity. Small 2012, 8, 362-366. (49)

Bengoechea, J. A.; Díaz, R.; Moriyón, I. Outer membrane differences

between pathogenic and environmental Yersinia enterocolitica biogroups probed with hydrophobic permeants and polycationic peptides. Infect. Immun. 1996, 64, 4891-4899. (50)

Zhu, X.; Shan, A.; Ma, Z.; Xu, W.; Wang, J.; Chou, S.; Cheng, B.

Bactericidal efficiency and modes of action of the novel antimicrobial peptide T9W against Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 2015, 59, 3008-3017. (51)

Dong, N.; Ma, Q.; Shan, A.; Lv, Y.; Hu, W.; Gu, Y.; Li, Y. Strand

length-dependent antimicrobial activity and membrane-active mechanism of arginine-

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and valine-rich β-hairpin-like antimicrobial peptides. Antimicrob. Agents Chemother. 2012, 56, 2994-3003. (52)

Kim, Y. S.; Ahn, C. B.; Je, J. Y. Anti-inflammatory action of high

molecular weight Mytilus edulis hydrolysates fraction in LPS-induced RAW264.7 macrophage via NF-κB and MAPK pathways. Food Chem. 2016, 202, 9-14.

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Fig. 1. Design of synthetic α-helical antimicrobial peptides based on the heptad repeat sequence model. (A) Molecular three-dimensional structure of Leu, Ala, Phe, Trp, and Arg. (B) The three-dimensional structure projection and Helical wheel projection (C) of the model sequence XRRXRRR-H-RRRXRRX, where X is the hydrophobic residue (Leu, Ala, Phe, or Trp), R is the Arg residue, and H is the linker (Trp). The hydrophobic residue, Arg, and the linker (Trp) are color coded as yellow, red, and green, respectively.

Fig. 2. The CD spectra of the synthetic peptides. The peptides were dissolved in 10 mM PBS (pH 7.4) (A) or 30 mM SDS (B). The mean residue ellipticity was plotted against the wavelength. The values from three scans were averaged for each sample, and the peptide concentrations were fixed at 150 µM, each sample was assessed independently three times. The results showed that the designed peptides displayed an α-coil structure formation in anionic hydrophobic environments (SDS).

Fig. 3. Hemolytic activity of the synthetic peptides against human red blood cells (hRBCs). Hemolysis was determined by monitoring the optical density (OD) at 570 nm. The hRBCs in PBS and 0.1% Triton X-100 were employed as negative and positive controls, respectively. The data shown are the means ± SEM of three independent experiments using two replicates. The results showed that the designed amphiphiles had a slightly concentration-dependent toxicity against hRBCs. Fig. 4. Effects of synthetic peptides on the fluorescent intensity variation of P. aeruginosa 27853 incubated with NPN. The fluorescence intensity variation was monitored at an excitation wavelength of 350 nm and an emission wavelength of 420 nm. “100%” represents the “% NPN uptake” of the positive control polymyxin B. The data shown are the ± SEM of three independent experiments using two replicates. The results showed that the designed amphiphiles effectively permeabilized the outer membrane of P. aeruginosa in a concentration-dependent manner.

Fig. 5. Effects of synthetic peptides on the fluorescent intensity variation of P. aeruginosa 27853 incubated with diSC3-5. The fluorescence intensity variation was monitored at an excitation wavelength of 622 nm and an emission wavelength of 670 nm. The data shown are the means ± SEM of three independent experiments using two replicates. The results showed that the designed peptides could induce the depolarization of the cytoplasmic membrane of P. aeruginosa effectively in a concentration-dependent manner.

Fig. 6. Membrane damage in P. aeruginosa 27853 cells treated with the synthetic peptides at their 1 × MICs for 30 min. Flow cytometry was used to determine the fluorescent intensity of PI (25 µg/mL). The control was processed without peptides. (A) No peptide (negative control, 4.7%); (B) 1 (93%); (C) 5 (57.0%); (D) 7 (96.1%); (E) 9 (99.5%); (F) 10 (99.3%). “100%” indicates that all of the cells were stained with PI and appeared fluorescent. The flow cytometry results further demonstrated that the designed peptides 8 and 9 damaged the integrity of the cell 35



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

membrane.

Fig. 7. SEM (A) and TEM (B) micrographs of P. aeruginosa 27853 cells treated with 9 and 10 at their 1 × MICs for 1 h. The control was processed without peptides. In figure 7A, as indicated by the arrows, significant membrane distortion, corrugation, and damage were observed on the surface of P. aeruginosa cells after treatment with the amphiphiles compared to the smooth surface of the control. In figure 7B, as indicated by the arrows, the complete collapse of the cytoplasmic membrane and the dispersion of the intracellular contents were observed after treatment with the amphiphiles.

Fig. 8 Confocal fluorescence microscopic images of P. aeruginosa 27853 cells treated with FITC-labeled 8, 9, and melittin at 1 × MICs for 30 min. Panels on the left, middle, and right represent laser-scanning, merged, and transmitted-light scanning images of bacterial cells, respectively. This result further suggested that 8 and 9 were able to bind the P. aeruginosa cells membrane effectively.

Fig. 9. Peptide binding affinity for LPS from P. aeruginosa 27853. (A) BODIPY-TR-cadaverine displacement. The fluorescence intensity was monitored at an excitation wavelength of 580 nm and an emission wavelength of 620 nm. The graphs were derived from the average values of three independent trials compared to results with melittin at the same concentration. AU, arbitrary units. The results showed that 8 and 9 displayed a concentration-dependent increase in the fluorescent intensities, demonstrating the abilities of the amphiphiles to bind LPS. (B, C, D) SPR spectroscopy of the interaction kinetics of the peptides and LPS. The peptides were immobilized on a CM5 sensor chip as a ligand, and LPS was diluted in a series of concentrations. The values were recorded in response unit (RU) for the indicated concentrations of LPS. (B) 8, (C) 9, (D) Melittin. These results illustrated that the RU of LPS binding to immobilised 8, 9, and melittin was enhanced rapidly and in an obvious dose-dependent pattern at concentrations of 3.125, 6.25, or 12.5 µg/mL.

Fig. 10. The inflammatory response induced by LPS from P. aeruginosa 27853 was inhibited by the peptides. RAW264.7 cells (5 × 105 cells/mL) were treated with 100 ng/mL LPS in the absence or presence of 10 mg/L the peptides for 18 h. (A) The inhibitory effects of peptides on LPS-stimulated nitric oxide (NO) production in RAW264.7 cells. The cell culture media were collected, and the amount of nitrite released within the media was measured by monitoring the absorbance at 540 nm using Griess reagent. The error bars represent the standard deviations of the mean determined from three independent experiments. (B) The TNF-α production in RAW264.7 cells. The levels of TNF-α in the cytoplasm were determined by ELISA using a commercial kit for mouse TNF-α. (C, D, E) Inflammatory cytokine expression in RAW264.7 cells. The cDNA was analyzed by qRT-PCR. The gene

36


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expression changes for IL-1β, IL-6, and TNF-α were expressed as the fold-change in the levels of transcription in treated cells compared to un-treated cells. (F) The key signal molecules of the NF-κB pathway were analyzed by Western blot. The proteins assessed include TLR4, IκB α, and iNOS and β-actin served as a protein loading control. The data were normalized to the level mGAPD, and three independent experiments in three duplicates were performed. The error bars represent mean ± SEM. Bars with different letters represent significantly different mean values with p < 0.01 (n = 3). These results showed that the optimized amphiphiles possessed well-defined anti-inflammatory properties, inhibited NO and the production of the pro-inflammatory factors TNF-α, IL-6, and IL-1β because of the direct scavenging and modulation of cellular inflammatory pathways, including TLR4, IκB α, and iNOS expression.

Fig. 11. (A) Formation of P. aeruginosa 27853 biofilms treated with peptides at their 1 ×, 4 ×, and 8 × MICs for 24 h. The biomass of a biofilm was assessed by crystal violet staining and quantified at 595 nm. The results were expressed as the percentage of biofilm formation with a maximal percentage corresponding to the control well in the absence of peptides. (B) SEM images of P. aeruginosa 27853 biofilms treated with peptides at their 4 × MICs for 1 h. The control was processed without peptide. All experiments were performed at least three times. These results provided direct evidence that the designed amphiphiles had the ability to kill bacteria embedded within biofilms and efficiently mediate the dispersion of biofilms.

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Page 38 of 53

Table 1. Peptide designs and their key physicochemical parameters.

peptide

Peptide

Sequence

Theoretical MW

Measured MWa

Charge

Hb

µHb

code/compound 1

LW

LRRLRRR W RRRLRRL-NH2

2218.74

2217.78

+11

-0.07

0.79

2

AW

ARRARRR W RRRARRA-NH2

2050.42

2049.45

+11

-0.44

0.47

3

FW

FRRFRRR W RRRFRRF-NH2

2354.81

2353.85

+11

-0.05

0.81

4

WW

WRRWRRR W RRRWRRW-NH2

2510.96

2509.99

+11

0.08

0.92

5

LrW

LRRLRRR W LRRLRRR-NH2

2218.74

2217.78

+11

-0.07

0.64

6

L2 W

(LRRLRRR)2

W

4233.25

4232.31

+21

-0.15

0.74

7

L3 W

W

6247.77

6246.84

+31

-0.18

0.70

8

LW2

LRRLRRR WW RRRLRRL-NH2

2404.95

2403.99

+11

0.08

0.47

9

LW3

LRRLRRR

2591.17

2590.21

+11

0.20

0.12

(RRRLRRL)2-NH2 (LRRLRRR)3 (RRRLRRL)3-NH2 WWW

RRRLRRL-NH2 a

The molecular weight (MW) was measured by mass spectroscopy (MS).

b

Hydrophobicity (H) and hydrophobicity moment (µH) were calculated online at:

http://heliquest.ipmc.cnrs.fr/cgi-bin/ComputParams.py.

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Table 2. Antibacterial and cytotoxicity of the synthetic peptides. Strains

MICa (mg/L) 1

2

3

4

5

6

7

8

9

Melittin

Ciprofloxacin

Gentamicin

Ceftazidime

E. coli ATCC 25922

16

64

8

32

>128

8

8

16

4

2

4

0.5

2

S. aureus ATCC 29213

16

64

16

16

64

16

8

8

8

8

4

1

16

P. aeruginosa ATCC 27853

16

128

8

4

32

8

16

8

4

4

4

2

2

P. aeruginosa CICC 10419

32

256

256

256

256

4

4

16

8

8

8

16

32


16

256

256

256

64

8

8

16

8

16

8

32

32


16

256

32

16

32

4

4

8

4

8

16

8

8

drug-resistant P. aeruginosa 11411

32

256

256

256

128

8

16

8

4

4

256

32

32

P. aeruginosa CICC11421

32

256

256

256

256

16

8

16

8

8

16

256

32

P. aeruginosa (ceftazidime-resistant) 11431

16

256

16

8

64

8

8

16

4

8

8

8

256

clinical isolates P. aeruginosa 21328

32

256

256

256

128

8

4

32

4

4

256

256

256

P. aeruginosa 25349

8

32

8

32

32

8

8

16

4

4

8

4

64

P. aeruginosa 26305

32

256

64

32

64

16

8

8

4

2

8

2

128

GMb

18.0

194.0

68.6

59.7

78.8

7.1

6.7

11.3

4.2

4.8

17.1

16.0

39.4

c

MHC

>256

>256

>256

>256

>256

64

64

256

>256

0.25

>256

>256

>256

Cytotoxicityd

256

>256

256

128

>256

128

64

128

256

0.5

>256

>256

>256

TIe

>14.2

>1.3

>3.7

>4.3

>3.2

9.0

9.6

22.7

>61.0

0.05

>15.0

>16.0

>6.5

a

Minimal inhibitory concentration (MIC), the bacterial strains used were:E. coli ATCC25922, S. aureus ATCC29213,P. aeruginosa ATCC27853, P. aeruginosa CICC10419, P. aeruginosa

CICC21625 and P. aeruginosa CICC21630, drug-resistant P. aeruginosa 11411 (ciprofloxacin-resistant), P. aeruginosa 11421 (gentamicin-resistant) and P. aeruginosa 11431(ceftazidime-resistant), clinical isolates P. aeruginosa 21328, P. aeruginosa 25349 and P. aeruginosa 26305. b

Geometric mean (GM) of MIC values for the P. aeruginosa strains tested.

c

The minimal hemolytic concentration (MHC) was the lowest hemolysis concentration that leaded to 10% hemolysis of human red blood cells (hRBCs).

d

The cytotoxicity was the lowest peptide concentration that reduced the activity of RAW 264.7 macrophages by 10%.

e

The therapeutic index (TI) was calculated as MHC/GM.

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Page 40 of 53

Table 3. Antimicrobial activities of the synthetic peptides in various salt environments. Peptides

MICa (mg/L) Controlb

NaClb

KClb

NH4Clb

ZnCl2b

MgCl2b

FeCl3b

8

8

16

8

8

8

32

8

9

4

8

4

4

8

16

4

Melittin

4

64

8

8

8

32

16

a

The minimum inhibitory concentration (MIC) was the lowest concentration of 8, 9, and melittin that inhibited

b

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

bacterial growth. P. aeruginosa 27853 was used as the reference strain.

mM, and 4 µM, respectively, and the control MIC values were determined under these physiological salts environments.

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89x60mm (300 x 300 DPI)



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79x45mm (600 x 600 DPI)


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80x45mm (600 x 600 DPI)



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70x35mm (600 x 600 DPI)


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21x11mm (600 x 600 DPI)


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149x99mm (300 x 300 DPI)



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40x18mm (300 x 300 DPI)


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90x90mm (300 x 300 DPI)



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158x87mm (300 x 300 DPI)


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209x202mm (150 x 150 DPI)



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107x111mm (300 x 300 DPI)


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160x80mm (300 x 300 DPI)


Novel Design of Heptad Amphiphiles To Enhance Cell Selectivity, Salt

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