Structure and Function of a Potent Lipopolysaccharide-Binding

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Structure and Function of A Potent Lipopolysaccharidebinding Antimicrobial and Anti-inflammatory Peptide Lin Wei, Juanjuan Yang, Xiaoqin He, Guoxiang Mo, Jing Hong, Xiuwen Yan, Donghai Lin, and Ren Lai J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/jm4004158 • Publication Date (Web): 17 Apr 2013 Downloaded from http://pubs.acs.org on April 27, 2013

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Journal of Medicinal Chemistry

Structure and Function of A Potent Lipopolysaccharide-binding Antimicrobial and Anti-inflammatory Peptide Lin Wei1¶, Juanjuan Yang2¶, Xiaoqin He1¶, Guoxiang Mo1, Jing Hong4, Xiuwen Yan1*, Donghai Lin2*, Ren Lai1, 3* 1

Key Laboratory of Microbiological Engineering of Agricultural Environment, Ministry of

Agriculture, Life Sciences College of Nanjing Agricultural University, Nanjing 210095, Jiangsu, China 2

Key Laboratory of Chemical Biology of Fujian Province, Department of Chemistry, College of

Chemistry and Chemical Engineering, Xiamen university, Xiamen 361005, Fujian, China 3

Key Laboratory of Animal Models and Human Disease Mechanisms, Kunming Institute of

Zoology, Chinese Academy of Sciences, Kunming 650223, Yunnan, China 4

College of Biological Science and Technology, Fuzhou University, Fuzhou 380108, Fujian,

China ABSTRACT Antimicrobial peptides (AMPs) play pivotal roles in the innate defense of vertebrates. A novel AMP (cathelicidin-PY) has been identified from the skin secretions of the frog Paa yunnanensis. Cathelicidin-PY has an amino acid sequence of RKCNFLCKLKEKLRTVITSHIDKVLRPQG. Nuclear Magnetic Resonance (NMR) spectroscopy analysis revealed that cathelicidin-PY adopts 1

ACS Paragon Plus Environment


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a tertiary structure with a mostly positively charged surface containing a helix (Thr15–Ser19). It possesses strong antimicrobial activity, low hemolytic activity, low cytotoxicity against RAW 264.7 cells and strong anti-inflammatory activity. The action of antimicrobial activity of Cathelicidin-PY is through the destruction of the cell membrane. Moreover, cathelicidin-PY exerts anti-inflammatory activitiy by inhibiting the production of nitric oxide (NO) and inflammatory cytokines such as tumor necrosis factor (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). Cathelicidin-PY inhibits the activation of Toll-like receptor 4 (TLR4) inflammatory response pathways induced by lipopolysaccharide (LPS). The NMR titration experiments indicated that cathelicidin-PY can bind to LPS. In conclusion, we have identified a novel potent peptide antibiotic with both antimicrobial and anti-inflammatory activities and laid the ground work for future research and development.

KEYWORDS:

Amphibian;

Antimicrobial

Peptide;

Cathelicidin;

Anti-inflammatory;

Lipopolysaccharide;

INTRODUCTION In vertebrates, cathelicidins and defensins play critical roles in innate immune system that can provide a first line of defense against various infectious factors over an entire lifespan.1-3 Defensins contain 3 or 4 disulfide bridges. Vertebrate defensins are classified into three families including α-, β-, and θ-defensins based on their cysteine motifs. Most cathelicidins contain none or one disulfide bridge. Cathelicidins are composed of a N-terminal signal peptide (about 30 residues), and a highly conserved cathelin domain (99–114 residues) followed by a C-terminal 2


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mature peptide (12–100 residues). The C-terminal mature peptides are characterized by remarkable structural diversity.4 The first cathelicidin named Bac5 was identified from bovine neutrophils. Subsequently, large quantities of cathelicidins have been identified from most vertebrates including fishes, birds, and mammals.2, 5, 6 The first reptile cathelicidin was identified from the snake venom of Bungarus fasciatus in our previous work.7 Recently, we have identified the first amphibian cathelicidin from the skin of the frog, Amolops loloensis.8 Many cathelicidin antimicrobial peptides exhibit strong antimicrobial property, high specificity amongst different bacteria, and weak cytotoxicity to mammalian cells, which make them excellent candidates for the development of antimicrobial agents. Many antimicrobial peptides such as Iseganan, Omiganan, and MBI 594AN in clinical trials are cathelicidins, suggesting that cathelicidin peptides are good templates for the development of peptide antibiotics.9 In order to identify potent cathelicidin antimicrobial peptides and to prove the widespread presence of cathelicidins in amphibians, we continue our work to identify and characterize cathelicidin antimicrobial peptides from amphibians. A novel cathelicidin (cathelicidin-PY) with unique structure was purified from the frog skin secretions of P. yunnanensis. A variety of biological and biophysical assays demonstrated that cathelicidin-PY possesses strong antimicrobial and anti-inflammatory activities and low cytotoxic ability against RAW 264.7 cells. Cathelicidin-PY’s bacteria-killing kinetics was also assessed. The tertiary structure of cathelicidin-PY was resolved by using NMR spectroscopy. The interactions between cathelicidin-PY and LPS were detected by NMR titration experiments to understand the mechanisms of its antimicrobial and anti-inflammatory activities. The effects of cathelicidin-PY 3



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on LPS-activated TLR4 signaling pathways were also exploited. EXPERIMENTAL SECTION Collection of Frog Skin Secretions Both adult sexes of P. yunnanensis (n = 30; weight range 40 - 60 g) were collected in Yunnan Province of China. Skin secretions were collected according to our previous method.10 Frogs were stimulated by volatilized anhydrous ether immersed in absorbent cotton. Copious secretions were released from the frog skin surface. Skin secretions were collected by washing frogs with 0.1 M NaCl solution (containing 1% (v/v) protease inhibitor cocktail, Sigma-Aldrich, P8340-5). The collected solutions (600 mL of total volume) were quickly centrifuged and the supernatants were lyophilized. All experiments were approved by Kunming Institute of Zoology, Chinese Academy of Sciences. Peptide Purification The aliquot (0.5 g) of lyophilized skin secretion was dissolved in 10 mL of 0.1 M phosphate buffer, pH 6.0 (PBS) and centrifuged at 5,000 G for 10 min. The supernatant was subjected to preliminary separation by a Sephadex G-50 (Superfine, Amersham Biosciences, 2.6 cm diameter, 100 cm length) gel filtration column equilibrated with PBS. Elution was performed with the same buffer, collecting in fractions of 3.0 mL. The eluted fractions (3 mL per fraction) were monitored at 280 nm and subjected to antimicrobial assays. The collected sample that possessed interesting activity was further purified by a C18 reversed-phase high performance liquid chromatography (RP-HPLC, Gemini C18 column, 5 µm particle size, 110 Å pore size, 250 mm length, 4.6 mm diameter) column. The elution was performed using a linear gradient of 0–80% 4


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acetonitrile containing 0.1% (v/v) trifluoroacetic acid in 0.1% (v/v) trifluoroacetic acid/water over 70 min as illustrated in Figure 1A. UV-absorbing peaks were collected, lyophilized, and assayed for antimicrobial activity. Primary Structural Analysis The purified peptide was subjected to automated Edman degradation analysis on an Applied Biosystems pulsed liquid-phase sequencer (model ABI 491). 0.5 µL of the purified peptide (in 0.1% (v/v) trifluoroacetic acid/water) was spotted onto a matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) plate with a 0.5 µL α-cyano-4-hydroxycinnamic acid matrix (10 mg/mL in 60% acetonitrile) and analyzed by an UltraFlex I mass spectrometer (Bruker Daltonics) in positive ion mode. Circular Dichroism Analysis Circular dichroism (CD) experiments were used to estimate the secondary structural elements of cathelicidin-PY and optimize the NMR buffer suitable for NMR experiments. CD spectra were recorded on a Jasco-810 spectropolarimeter (Jasco, Tokyo, Japan) with a 1-mm path-length cell (25°C, 0.2-nm interval from 190 to 260 nm). Samples were prepared in H2O, membrane-mimetic environments (TFE/H2O and SDS/H2O solution), NaCl solution, Na2HPO4-NaH2PO4 buffer, respectively. The peptide concentration was 0.2 mg/ml for all CD experiments. For each spectrum, the data from 3 scans were averaged and smoothed using the Jasco-810 software. CD data were expressed as the mean residue ellipticity (θ) in deg.cm2.dmol-1. NMR Spectroscopy Samples for NMR experiments contained 0.3 ml of 4.38 mM cathelicidin-PY in 95% H2O/ 5



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TFE (6:4, v/v), 5% D2O at pH 6.2, 25°C. All NMR spectra were recorded on a Bruker Avance III 600 MHz spectrometer. Structure calculation of the peptide was performed according to the standard ARIA/CNS protocol.11-14 The 1H spin systems of the individual amino acid residues were identified from 2D 1H-1H COSY spectrum (number of scans 16, data points collected 2048×512) and 1H-1H TOCSY spectrum (number of scans 32, data points collected 2048×256). Proton-proton distance restraints were derived primarily from the NOESY spectrum (number of scans 24, data points collected 2048×256) recorded with a mixing time of 300 ms. Final structures were analyzed by using the program packages MOLMOL15 and PROCHECK.16 cDNA Library Construction and Screening Total RNA was extracted from the skin of both adult sexes of P. yunnanensis using RNeasy Protect Mini Kit (QIAGEN, Germany). SMARTTM cDNA Library Construction Kit (Clontech, United States/Canada) was used to prepare cDNA. Two primers provided by the manufacturer (CDS III/3’ PCR Primer, 5’-ATTCTAGAGGCCGAGGCGGCCGACATG-d(T)30N–1N-3’ (N = A, G,

C,

or

T;

N–1

=

A,

G,

or

C),

and

SMART

Ⅳ

oligonucleotide,

5’-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3’) were used for the first strand synthesis. 5’ PCR Primer (5’-AAGCAGTGGTATCAACGCAGAGT-3’) provided by the manufacturer was used for the synthesis of the second strand. A directional cDNA library was constructed with a plasmid cloning kit (SuperScriptTM Plasmid System, GIBCO/BRL) following the instructions of manufacturer, producing a library of about 2.9 × 105 independent colonies. A PCR-based method was used for screening and isolating the clones from the cDNA library.

Two

oligonucleotide 6


primers,

S1

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(5’-A(A/G)(C/T)TT(A/G)CA(A/C/G/T)A(A/G)(A/G)AA(A/G)TT(A/G)CA(C/T)T-3’, according to the sequence determined by Edman degradation, in the antisense direction) and 5’ PCR primer as mentioned above (in the sense direction) were used in the PCR reactions. The full length cDNA was finally obtained from primers of S2 (5’-ATGAAGGTCTGGCAGTGTGTG-3’, sense primer) and 3’ PCR primer (antisense primer) from the library kit. The PCR conditions were, 4 min at 95 °C, followed by 28 cycles of 30 sec at 95 °C, 30 sec at 55 °C, 40 sec at 72 °C, and concluded by 10 min extension at 72 °C. DNA sequencing was performed on an Applied Biosystems DNA sequencer, model ABI PRISM 377. Phylogenetic Analysis Cathelicidin sequences were obtained from the protein database at the National Center for Biotechnology Information (NCBI). The ClustalX program (version 1.81) and the Molecular Evolutionary Genetics Analysis (MEGA) software (version 5.0) were used to construct phylogenetic tree by neighbor-joining analysis.17, 18 Expression Profile of Tissues The

gene

expression

profile

of

cathelicidin-PY

was

analyzed

by

reverse

transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from different tissues using RNeasy Protect Mini Kit (QIAGEN, Germany) according to the manufacturer’s instruction. Equal amounts of total RNA was used in the first-strand cDNA synthesis using a PrimeScript®

RT-PCR

Kit

(Takara).

The

(5’-ATGAAGGTCTGGCAGTGTGTGCT-3’,

specific

primers

sense),

and

were

5’-cathelicidin 3’-cathelicidin

(5’-AGATGTAGCTGAAGGATCCACGT-3’, antisense). The control PCR primers were, 5’-actin 7



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(5’-ACCACAGCAGAAAGAGAAATCGT-3’,

sense),

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and

3’-actin

(5’-ATCCAGACGGAGTATTTGCGCT-3’, antisense). All PCR analyses were conducted at 95°C for 3 min, followed by 30 cycles of 95 °C (30 s), 56 °C (30 s), 72 °C (3 min), and cocluded by a 10 min extension period at 72 °C. Antimicrobial Testing and Bacteria Killing Kinetics Antimicrobial activity of cathelicidin-PY was tested according to our previous methods.10 In vitro bacteria killing kinetics of cathelicidin-PY and the antibiotics levofloxacin hydrochloride and sodium chloride injection(LHSC, its minimal inhibitory concentration (MIC) for Escherichia coli ATCC 25922 is 0.05 µg/mL) against E. coli ATCC 25922 were determined according to the methods described by Mygind et al.19 Scanning Electron Microscopy (SEM) SEM was performed to exploit the effects of cathelicidin-PY on the membrane morphology of the bacteria E. coli ATCC 25922 and clinically isolated E. coli 08A852. Bacteria (approximately 2 × 107 CFU/mL) were cultured with cathelicidin-PY (1 × MIC) at 37 °C for 30 min. The culture was centrifuged at 1,000 G for 10 min and the pellet was fixed with 2.5% buffered glutaraldehyde at 4 °C for 2 h. The bacteria were then postfixed in 1% buffered osmium tetroxide for 2 h, dehydrated in a graded series of ethanol, frozen in liquid nitrogen cooled tertbutyl alcohol and vacuum dried overnight. After mounting onto aluminum stubs and vacuum sputter-coating with gold, the sample was analyzed with a Hitachi S-3000N SEM following the manufacturer’s instruction. Hemolysis and Cytotoxicity 8


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Hemolytic activity was evaluated by incubating the tested samples with human red blood cells to check hemoglobin concentration by monitoring the absorbance at 540 nm, using 1% Triton X-100 as a positive control. Cytotoxicity against RAW 264.7 murine macrophage cells was determined according to the methods described by Mygind et al.19 Regulation of Nitrite Production Nitrite accumulation in culture medium is a reflection of NO production.20-22 Lipopolysaccharide (LPS) are also called endotoxins, which possess strong immunogenicity and can provoke a immune response in immunocytes. RAW 264.7 murine macrophage cells (1 × 107) cultured in RPMI-1640 (containing 10% fetal bovine serum, 100 U/mL penicillin and 100 µg/mL streptomycin, Invitrogen) were plated and adhered to a 96-well culture plate. After the cells were treated with or without cathelicidin-PY (2.5, 5, 10, and 20 µg/mL) and/or LPS (100 ng/mL, from Escherichia coli 055:B5, Sigma-Aldrich, USA) for 24 h, culture medium was harvested to detect the nitrite level using Griess reagent (Sigma-Aldrich, USA) according to the manufacturer’s instructions. The absorbance at 540 nm was measured on a microplate reader (Epoch Etock, BioTek, USA), and nitrite accumulation level was deduced from the standard curve generated with NaNO2 (Sigma-Aldrich, USA). Regulation of Inflammatory Cytokine Production RAW 264.7 murine macrophage cells (2 × 105) were plated and adhered to a 96-well culture plate. After the cells were treated with or without cathelicidin-PY (2.5, 5, 10, and 20 µg/mL) and/or LPS (20 ng/mL, from Escherichia coli 055:B5, Sigma-Aldrich, USA) for 24 h, supernatants of the culture medium were harvested for TNF-α, IL-6 and MCP-1 analyses using 9



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the commercially enzyme-linked immunosorbent assay (ELISA) kits. Mouse TNF-α, IL-6 and MCP-1 ELISA kits (DAKAWE, Beijing, China) were used in this experiment. The absorbance at 450 nm was measured on a microplate reader (Epoch Etock, BioTek, USA). Western Blot Analysis RAW 264.7 murine macrophage cells (1 × 106/well) were plated to a 24-well culture plate and transferred to serum-free RPMI-1640 for a 16-h incubation. The cells were pretreated with cathelicidin-PY (0, 5, 10, and 20 µg/mL) for 1 h before the addition of LPS (0, 100 ng/mL, from Escherichia coli 055:B5, Sigma-Aldrich, USA). After incubation for 3 h, the cells were collected by centrifugation (1, 000 g for 5 min) and washed twice with ice-cold phosphate-buffered saline (PBS) (8 g/L NaCl, 0.2 g/L KCl, 0.2 g/L KH2PO4, 2.89 g/L Na2HPO4·12H2O, pH 7.4). The washed cell pellets were then used to extract cytoplasmic or nuclear proteins for western blot analysis according to our previous methods.23 Primary antibodies against β-actin (1:5000, Santa Cruz Biotechnology, USA) and TLR4, JNK, NF-κB p65 (1:2000; Cell Signaling Technology, Beverly, MA, USA) were used in western blot analysis. NMR Titration The interaction between cathelicidin-PY and LPS was detected by using NMR titration experiments. TOCSY spectra were recorded on the cathelicidin-PY solution (2.5 mM, in 95% H2O/TFE-d3 (6:4, v/v), 5% D2O at pH 6.2, 25 °C, 500 µl) with or without LPS (1 or 5 µg/ml, from Escherichia coli 055:B5, Sigma, USA). The peak widths of TOCSY spectra were used to monitor peak broadening during the LPS titration process, which is usually used for the case of intermediate conformational exchange. 10


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Synthetic Peptides All synthetic peptides used were synthesized by GL Biochem (Shanghai) Ltd. (Shanghai, China) and analyzed by HPLC and mass spectrometry to confirm a purity higher than 98%. RESULTS Purification of Cathelicindin-PY The skin secretions of P. yunnanensis were separated into seven fractions by the Sephadex G-50 gel filtration as illustrated in Figure 1A. The fraction that contained antimicrobial activity marked by an arrow was pooled and applied to a C18 RP-HPLC column for further purification (shown in Figure 1B). The purified antimicrobial peptide (named cathelicidin-PY) was indicated by an arrow in Figure 1B. Its molecular weight and purity were analyzed by MALDI-TOF mass spectrometry (MS) (Figure 1C). Structural Characterization The purified antimicrobial peptide was subjected to amino acid sequence analysis by automated

Edman

degradation.

Its

amino

acid

sequence

was

determined

as

RKCNFLCKLKEKLRTVITSHIDKVLRPQG, composed of 29 amino acid residues including two

cysteines,

which

could

potentially

form

an

intra-molecular

disulfide

bridge.

MALDI-TOF-MS gave an observed molecular weight of 3423.4 Da (Figure 1C), which matches well with its theoretical molecular weight (3423.2 Da). The presence of an intra-molecular disulfide bridge in native cathelicidin-PY was further confirmed by a synthesized peptide, which had the same observed molecular weight and RP-HPLC elution manner with the native cathelicidin-PY. cDNA Cloning

11



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Upon screening of the skin cDNA library, several clones containing inserts of around 600-base pairs, were identified and isolated. The complete nucleotide sequence of cathelicidin-PY (GenBank accession number JQ836655) and the deduced amino acid sequence are shown in Figure 2. The gene that encoded cathelicidin-PY was proved to contain a coding region of 456 nucleotides. The encoded amino acid sequence corresponds to a polypeptide of 152 amino acids including mature cathelicidin-PY. BLAST comparisons showed that the precursor is a member of the cathelicidin family containing a conserved cathelin domain and a variable C-terminus. Expression profile analysis indicated that mRNA of cathelicidin-PY was expressed in all of the tissues (Figure S1). Phylogenetic Analysis Most known cathelicidins including cathelicidin-PY were used for phylogenetic analysis. Evolutionary analysis demonstrated that all vertebrate cathelicidins form two distinct clusters with fish cathelicidins located in a separated clade from others. All cathelicidins from quadrupeds are in the second cluster (Figure 3). This cluster of quadrupeds is again divided into two major groups. Two cathelicidins from amphibians are coordinated by avian, snake, and the most divergent mammalian cathelicidins. This evolution analysis indicated that amphibian cathelicidins form a link that predate reptiles but postdate fishes. Solution Structure The secondary structural components of cathelicidin-PY in different solutions are exhibited in Table 1 and Figure S2. The CD spectra of cathelicidin-PY dissolved in H2O showed a strong negative peak at 199 nm, indicating that cathelicidin-PY adopted a random-coil conformation (Figure S2A). In the membrane-like environments of either TFE/H2O or SDS/H2O solutions, the 12


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CD spectra showed double negative peaks at 208 nm and 222 nm, indicative of the main secondary structure component of α-helix (Figure S2A & B). Furthermore, in either NaCl (0.9%, w/v, Figure S2C) or Na2HPO4-NaH2PO4 (20 mM, pH 7.4, Figure S2D), cathelicidin-PY also adopted a random-coil conformation. Proton resonances of almost all residues of cathelicidin-PY were assigned in the COSY, TOCSY (Figure 8A) and NOESY (Figure 8B) spectra (Table S1. BMRB accession number, 18350). All the long-range NOEs used in structure calculation are listed in Table S2. Backbone atomic coordinate superposition of the 20 lowest energy structures of cathelicidin-PY is illustrated in Figure 4A (PDB ID: 2LR7). Structural statistics for the 20 lowest energy structures of cathelicidin-PY is shown in Table S3. The average RMSD value of the 20 lowest energy structures is 0.40 Å for backbone atoms and 1.09 Å for heavy atoms in secondary structures. No NOE violation above 0.3 Å exists in these structures. The Ramachandran plot reveals that the distributions of the backbone dihedral angles (φ, ψ) within the whole structures of the ensemble are 52.3% of the residues in the most favored regions, 43.8% in additionally allowed regions, 3.8% in generously allowed regions, no residues in disallowed regions. The tertiary structure of cathelicidin-PY contains a helix (residues Thr15-Ser19, Figure 4C) which is stabilized by one intramolecular disulfide bond between Cys3-Cys7 and two hydrogen bonds between Phe5-Arg26, Lys10-Arg14 (Figure 4B). The secondary structure component of helix contained in the NMR structure of cathelicidin-PY was supported both by the secondary structure prediction based on the CD spectrum and by the Hα CSI prediction (data not shown). Most regions of the electrostatic surface of cathelicidin-PY are positively charged (Figure 4D). 13



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Antimicrobial Activities As listed in Table 2, cathelicidin-PY exerted strong antimicrobial abilities (MIC ranging from 4.69-37.5 µg/mL) against most of the tested microorganisms except B. subtilis ATCC 6633 (MIC > 100 µg/mL). It showed the same antimicrobial activity against both clinically isolated drug-resistant and standard strains. Cathelicidin-PY displayed variable antimicrobial activity in different salt solutions as listed in Table 3. Bacteria-killing Kinetics Using the antibiotics levofloxacin hydrochloride and sodium chloride injection (LHSC) as a positive control, antibacterial property of cathelicidin-PY was tested by using a colony counting assay. Cathelicidin-PY rapidly exerted its antibacterial properties in 1 ×, 5 ×, and 10 × MICs as listed in Table 4. It took less than 40 minutes to kill all the E. coli ATCC 25922 at one time of MIC. At the concentration of 5 and 10 times of MIC, cathelicidin-PY just took less than 20 and 10 minutes to clear the bacterium, respectively. The antibacterial activity proved to be lethal for E. coli ATCC 25922 because it was not capable of resuming growth on agar plates. In contrast, the antibiotics, LHSC took at least 60 min to completely kill the bacteria at 5 × MIC. Scanning Electron Microscopy The effects of cathelicidin-PY on the membrane morphology of E. coli were investigated by SEM as illustrated in Figure 5. For both standard and drug-resistant strains of E. coli, there are clear morphology differences between cathelicidin-PY-treated bacteria and the control. Their outer membranes of untreated E. coli were long, rod-shaped, and smooth (Figure 5A & 5C). There were breaks in the plasma membranes of cathelicidin-PY-treated bacteria, and intracellular 14


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inclusions had flowed out of the membranes. In addition, obvious cell swell was observed in the treated bacteria (Figure 5B & 5D). Under SEM, normal E. coli is dark with high density and clear membrane boundary while the cathelicidin-PY-treated E. coli appeared light and had a vague cell boundary. Hemolysis and Cytotoxicity Human red blood cells were used to evaluate the hemolytic activity of cathelicidin-PY. Cathelicidin-PY induced little hemolytic activity in a dose-dependent manner. At a concentration of 12.5, 25, and 50 µg/mL, cathelicidin-PY induced 2.5, 3.5, and 5.5% human red cell hemolysis, respectively. Its cytotoxicity was evaluated by using RAW 264.7 murine macrophage cells. Cathelicidin-PY showed no cytotoxicity at concentrations lower than 25 µg/mL while it showed a little cytotoxicity at concentrations higher than 25 µg/mL. At a concentration of 50, 100, and 200 µg/mL, cathelicidin-PY induced 2.7, 3.3, and 5.1% death of RAW 264.7 murine macrophage cells, respectively. Inhibition of NO Production Induced by LPS As illustrated in Figure 6A, 100 ng/mL LPS induced 3.8 µg/mL nitrite release. Cathelicidin-PY inhibited nitrite production in a dose-dependent manner. At the concentration of 2.5, 5, 10, and 20 µg/mL, cathelicidin-PY inhibited about 17, 30, 50, and 70% of the nitrite production induced by LPS, respectively. Inhibition of Inflammatory Cytokines Production Induced by LPS The effects of synthesized cathelicidin-PY on the secretion of several inflammatory factors including TNF-α, IL-6, and MCP-1 induced by LPS in RAW 264.7 murine macrophage cells 15



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were detected as illustrated in Figure 6B-D. 20 ng/mL LPS alone induced the secretion of TNF-α (about 2500 pg/mL), IL-6 (about 550 pg/mL), and MCP-1 (about 1240 pg/mL), respectively. Cathelicidin-PY inhibited the secretion of these three inflammatory cytokines in a dose-dependent manner. At the concentration of 20 µg/mL, cathelicidin-PY inhibited LPS-induced TNF-α, IL-6, and MCP-1 production by 60, 86, and 68%, respectively. Inhibition of the Activation of LPS-Induced TLR4 Inflammatory Response Pathways LPS significantly induced activation of TLR4 expression and its downstream effectors, JNK sub-group of MAPK signal pathway and transcription factor NF-κB as illustrated in Figure 7. Cathelicidin-PY inhibited the expression of TLR4, JNK, and the translocation of NF-κB from cytoplasm to nucleus in a dose-dependent manner. At the concentration of 20 µg/mL, cathelicidin-PY inhibited 85% TLR4 expression and 74% NF-κB translocation induced by 100 ng/mL LPS, respectively. JNK expression induced by LPS was also inhibited by this peptide. JNK2 expression induced by 100 ng/mL LPS was completely blocked by 10 or 20 µg/mL cathelicidin-PY. Cathelicidin-PY Binds to LPS NMR titration was performed to investigate the interaction between cathelicidin-PY and LPS. Previous works demonstrated that LPS with negative charges were usually bound to peptides or proteins via electrostatic interaction.24 TOCSY spectra were used to monitor peak broadening during the LPS titration process, in which peak widths of some resonances broadened or even disappeared as shown in Figure 9. This observation indicates that LPS was bound to cathelicidin-PY and the dissociate constant Kd is about µM (medium affinity). 16


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DISCUSSION AND CONCLUSION The skin of amphibians serve multiple functions including defense, respiration, and water regulation. They play key roles in the everyday survival of amphibians and their ability to thrive in a wide range of habitats and ecological conditions.25 Naked amphibian skins are susceptible to infection by microorganisms. Gene-encoded antimicrobial peptides are the main components of the biochemical arsenal designed to defend against infection by microorganisms in amphibian skins. The diversity of antimicrobial peptides discovered in amphibians is wide. More than 600 antimicrobial peptides belonging to more than 30 families have been identified from amphibians. However, little information is available about amphibian cathelicidin antimicrobial peptide even though some amphibian genomes have been sequenced. Until present, only one cathelicidin antimicrobial peptide had been identified from an amphibians.8 A novel antimicrobial peptide (cathelicidin-PY) exhibiting strong antimicrobial property (Table 2 & Table 3) with a unique structure was identified from the frog skin secretions of P. yunnanensis.

Its

primary

structure

was

determined

as

RKCNFLCKLKEKLRTVITSHIDKVLRPQG. cDNA cloning indicated that cathelicidin-PY is derived from a cathelicidin precursor containing 152 residues, indicating that cathelicidin-PY is a cathelicidin antimicrobial peptide although it shares no similarity to known peptides or proteins. Two cysteines in this sequence form an intra-molecular disulfide bridge. The structure motif is different from any other known cathelicidins. Most of cathelicidins are linear peptides that do not contain

cysteine.

(RLCRIVVIRVCR),

Only

several oadode

loop-structured

cathelcidins

(RYCRIIFLRVCR),4 17


including and

dodecapeptide protegrins


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

(RGGRLCYCRRRFCVCVGR) contain 2 or 4 cysteines,26,

Page 18 of 41

27

which formed 1 or 2

intra-molecular disulfide bridges. In contrastwith the aforementioned loop-structured cathelicidins, in which most of the residues are embraced in the disulfide loops, the disulfide loop in cathelicidin-PY is only composed of 5 residues. Most residues in cathelicidin-PY are extended in the C-terminal of the loop. The structural character of cathelicidin-PY is further confirmed by its tertiary structure, which is stabilized by one intramolecular disulfide bond between Cys3-Cys7 and two hydrogen bonds (Phe5-Arg26 and Lys10-Arg14) (Figure 4B). These results indicated that cathelicidin-PY has distinctive primary and tertiary structures. Combined with the previously identified amphibian cathelicidin,8 there are a total of two amphibian cathelicidins. Most of the known cathelicidin precursors were used for phylogenetic analysis. The phylogenetic tree constructed in this study suggests that in the course of evolution, amphibian cathelicidins play the role of a connecting bridge, predating reptilian but postdating fish cathelicidin. A significant coincidence is that all cathelicidins from quadruped animals are clustered together and fish cathelicidins are clustered separately in the phylogenetic tree (Figure 3). SEM analysis indicated that the bactericidal action of cathelicidin-PY is attributable to perturbation of the bacterial cell membrane (Figure 5). Many antimicrobial peptides are positively charged. They bind strongly and permeate into negatively charged bacterial phospholipid membranes.10, 28 Cathelicidin-PY has a net charge of +6 and possibly interacts with bacterial membranes. The observations from the tertiary structure elucidated that most regions of the electrostatic surface of cathelicidin-PY are positively charged (Figure 4D) and containing an 18


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amphiphilic structure of helix (Thr15–Ser19, Figure 4C), which allow cathelicidin-PY to bind to negatively charged

and

amphiphilic bacterial

membranes.

The

interaction

between

cathelicidn-PY and bacterial membranes was further investigated by monitoring the superposition of the TOCSY spectra of free cathelicidin-PY and in complex with LPS through NMR titration. It has been detected that most residues were involved in peak broadening after LPS addition (Figure 9). These results imply that the positively charged cathelicidin-PY binds to negatively charged LPS mostly via electrostatic interaction and the amphiphilic structure of helix.24 LPS binds to surface TLR4 molecules, triggering the secretion of various inflammatory factors, which contribute to the pathophysiology of septic shock and other immune diseases.29, 31 In addition, LPS also induces the expression of TLR4.29 As illustrated in Figure 6, LPS induced the secretion of inflammatory cytokines including TNF-α, IL-6, and MCP-1, which were significantly inhibited by cathelicidin-PY. This suggests that this peptide can act as an anti-inflammatory agent. To address the mechanism of inhibiting inflammatory factors secretion by cathelicidin-PY, the effect of cathelicidin-PY on TLR4 expression induced by LPS was exploited as illustrated in Fig. 7. LPS-induced TLR4 expression was significantly inhibited by cathelicidin-PY. The expression of two downstream molecules including JNK and NF-κB triggered by TLR4 were also significantly inhibited by cathelicidin-PY. Cathelicidin-PY can bind to LPS as mentioned above (Figure 9). These results suggest that cathelicidin-PY binds to LPS to block the induction of TLR4 expression, which in turn inhibits the expression of JNK and translocation of NF-κB. 19



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In conclusion, the identification and characterization of a novel cathelicidin (cathelicidin-PY) with a unique structure from the frog skin secretions of P. yunnanensis was reported. Cathelicidin-PY exhibited strong antimicrobial activity against both standard and clinically isolated drug-resistant microorganism strains. It was demonstrated to be a potent peptide antibiotic with antibacterial activities as well as anti-inflammatory activities. It did not exhibit significant cytotoxicity toward mammalian cells. Cathelicidin-PY could rapidly kill bacteria and neutralize endotoxins. All of these properties make cathelicidin-PY an excellent candidate for antibiotic development.

20


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FIGURE LEGENDS Figure 1. Purification of cathelicind-PY from the skin secretions of P. yunnanensis. (A) Sephadex G-50 gel filtration of skin secretions of P. yunnanensis. The fraction containing antimicrobial activity is marked by an arrow. (B) The fraction containing antimicrobial activity from the Sephadex G-50 gel filtration was further purified by C18 RP-HPLC column. The elution was performed with the indicated gradient of acetonitrile at a flow rate of 0.7 mL/min. The purified cathelicidin-PY is indicated by an arrow. (C) MALDI-TOF MS analysis of the purified cathelicidin-PY. Figure 2. The nucleotide sequence encoding cathelicind-PY and the deduced amino acid sequence of the precursor polypeptide. The sequence of mature cathelicidin-PY is boxed. The asterisk (*) indicates the stop codon. Figure 3. Evolutionary relationships of cathelicidin-PY and other cathelicidins in vertebrate. The phylogenetic dendrogram is a condensed tree based on Neighbor-Joining and p-distance methods. Only bootstrap values >50% (1000 replicates) are shown next to the branches. Cathelicidin-PY is marked with a triangle (▲). Figure 4. NMR structure of cathelicidin-PY in in 95% H2O/TFE-d3, 5% D2O. (6:4, v/v, pH 6.2, 25°C), (PDB ID Code: 2LR7). (A) Backbone superimposition of the 20 lowest energy structures. (B) The mean structure calculated from the 20 lowest-energy structures highlighting one disulfide bond (black solid line) and two hydrogen bonds (green broken lines). (C) Ribbon representation of the mean structure of cathelicidin-PY. (D) Electrostatic surface of cathelicidin-PY which takes the same orientation as that in the Panel A. Positively charged region and negatively charged region are shown in blue and red, respectively. The electrostatic surface was calculated and colored using the program MOLMOL.

21



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Page 22 of 41

Figure 5. Effects of cathelicidin-PY on membrane morphology of E. coli by scanning electron microscopy (SEM). A and B, E. coli ATCC 25922. C and D, clinically isolated drug-resistant E. coli 08A852. A and C, control. B and D, treated with cathlicidin-PY (1 × MIC). The breaks of membranes and the flowed out-intracellular inclusions are marked by arrows. Figure 6. Effects of cathelicidin-PY on nitrite and inflammatory cytokines production induced by LPS. (A) Nitrite. (B) TNF-α. (C) IL-6. (D) MCP-1. Control, only treated with LPS. Blank, without cathelicidin-PY and LPS. Data are mean ± SEM value of three separate experiments. *P < 0.05, **P < 0.01 significantly different compared to the control. Figure 7. Effects of cathelicidin-PY on inflammatory response signaling pathways. Raw 264.7 macrophage cells were treated with or without cathelicidin-PY and/or LPS as the indicated concentration. *P < 0.05, **P < 0.01 significantly different compared to the control (100 ng/mL LPS treated only). Figure 8. Proton spectra of free cathelicidin-PY in presence of membrane-like environment H2O/TFE-d3 (6:4, v/v) (25 °C, pH 6.2, ppm) (A) the TOCSY spectra of cathelicidin-PY. (B) the NOESY connectivity diagram for sequential assignments linked by dαN (the positions of K2 HA -C3 H and N4 HA -F5 H cross peaks are located below the diagonal). Figure 9. Interaction between cathelicidin-PY and LPS detected by NMR titration. (A) Superposition of the TOCSY spectra of cathelicidin-PY free and in the complex with LPS. The ratio of cathelicidin-PY to LPS was 2.5 mM : 5 µg. (B) Column diagram displays the relative peak heights (ratios of the peak heights of amide protons of free peptide to those in the complex with LPS), excluding Asn4, Phe5 (no signals in TOCSY spectra) and Pro27 (without NH) during the LPS titration process. The ratio of cathelicidin-PY to LPS was 2.5 mM : 1 µg and 2.5 mM : 5 µg.

22


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ASSOCIATED CONTENT Supporting Information Available Detailed materials and methods for antimicrobial testing, bacteria killing kinetics, hemolysis, cytotoxicity, and western blot analysis of cathelicidin-PY. Table S1, Chemical shifts of cathelicidin-PY in

H2O/TFE-d3 (6:4, v/v) (25 °C, pH 6.2, ppm). Table S2. All the long-range

NOEs used in structure calculation. Table S3, Structural statistics for the 20 lowest energy structures of cathelicidin-PY. Figure S1, Analysis of gene expression profile of cathelicidin-PY in different tissues by RT-PCR using gene-specific primers and actin as control. Figure S2, Circular dichroism spectra graphs of cathelicidin-PY in different solutions. (A) TFE/H2O. (B) SDS/ H2O. (C) 0.9% NaCl. (D) 20 mM Na2HPO4-NaH2PO4, pH 7.4. This material is available free of charge via the Internet at http://pubs.acs.org. PDB ID Code PDB ID Code: 2LR7 AUTHOR INFORMATION Corresponding Author *

Dr. Xiuwen Yan, Tel: +86-25-84396849. Fax: +86-25-84396542. E-mail: [email protected] .

*

Dr. Donghai Lin, Tel: +86-592-2186078. Fax: +86-592-2186078. E-mail: [email protected].

*

Dr. Ren Lai, Tel: Tel: +86-25-84396849. Fax: +86-25-84396542. [email protected].

Author Contributions ¶

These authors contributed equally to this paper.

The manuscript was written through contributions of all authors. All authors have given approval 23



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Page 24 of 41

to the final version of the manuscript. All the authors declare that there is no conflict of interest in this work. ACKNOWLEDGM ENT We gratefully acknowledge Jeff Jinfeng Bai (Shanghai American School) for spelling revisions. This work was supported by Chinese National Natural Science Foundation (30830021, 31025025, 31070701, 31000960, 31025025, U1132601, 31000335, 31170717), the Ministry of Science and Technology (2010CB529800, 2009ZX09103-1/091, 2011ZX09102-002-10), the Ministry of Agriculture (2009ZX08009-159B), Jiangsu Province (BK2012365 and BE2012748), Yunnan Province Y103951111, and Nanjing Agricultural University (KJ2012023). ABBREVIATIONS USED AMP: antimicrobial peptide; MALDI-TOF: matrix-assisted laser desorption ionization time-of-flight; CD: circular dichroism; NMR: nuclear magnetic resonance; SEM: scanning electron microscopy; TLR: toll-like receptor. REFERENCES (1) Ramanathan, B.; Davis, E. G.; Ross, C. R.; Blecha, F. Cathelicidins: microbicidal activity, mechanisms of action, and roles in innate immunity. Microb. Infect. 2002, 4, 361–372. (2) Zaiou, M.; Gallo, R. L. Cathelicidins, essential gene-encoded mammalian antibiotics. J. Mol. Med. 2002, 80, 549–561. (3) Zanetti, M. The role of cathelicidins in the innate host defenses of mammals. Curr. Issues Mol. Biol. 2005, 7, 179–196. (4) Zanetti, M.; Gennaro, R.; Scocchi, M.; Skerlavaj, B. Structure and biology of cathelicidins. 24


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Adv. Exp. Med. Biol. 2000, 479, 203-218. (5) Turner, J.; Cho, Y.; Dinh, N. N.; Waring, A. J.; Lehrer, R. I. Activities of LL-37, a cathelin-associated antimicrobial peptide of human neutrophils. Antimicrob. Agents Chemother. 1998, 42, 2206-2214. (6) Xiao, Y.; Cai, Y.; Bommineni, Y. R.; Fernando, S. C.; Prakash, O.; Gilliland, S. E.; Zhang, G.; Identification and functional characterization of three chicken cathelicidins with potent antimicrobial activity. J. Biol. Chem. 2006, 281, 2858-2867. (7) Wang, Y.; Hong, J.; Liu; X., Yang, H.; Liu, R.; Wu, J.; Wang, A.; Lin, D.; Lai, R. Snake cathelicidin from Bungarus fasciatus is a potent peptide antibiotics. PLoS One. 2008, 3, e3217. (8) Hao, X.; Yang, H.; Wei, L.; Yang, S.; Zhu, W.; Ma, D.; Yu, H.; Lai, R. Amphibian cathelicidin fills the evolutionary gap of cathelicidin in vertebrate. Amino Acids. 2012, 43, 677-685. (9) Gordon, Y. J.; Romanowski, E. G.; McDermott, A. M. A review of antimicrobial peptides and their therapeutic potential as anti-infective drugs. Curr. Eye. Res. 2005, 30, 505-515. (10) Li, J.; Xu, X.; Xu, C.; Zhou, W.; Zhang, K.; Yu, H.; Zhang, Y.; Zheng, Y. T.; Lai, R. Anti-infection peptidomics of amphibian skin. Mol. Cell. Proteomics. 2007, 6, 882-894. (11) Delaglio, F.; Grzesiek, S.; Vuister, G. W.; Zhu, G.; Pfeifer, J.; Bax, A. NMRPipe: a multidimensional spectral processing system based on UNIX pipes. J. Biomol. NMR. 1995, 6, 277–293. (12) Nilges, M.; Macias, M. J.; O’Donoghue, S. I.; Oschkinat, H. Automated NOESY interpretation with ambiguous distance restraints: the refined NMR solution structure of the 25



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pleckstrin homology domain from beta-spectrin. J. Mol. Biol. 1997, 269, 408–422. (13) Rieping, W.; Habeck, M.; Bardiaux, B.; Bernard, A.; Malliavin, T. E.; Nilges, M. ARIA2: automated NOE assignment and data integration in NMR structure calculation. Bioinformatics. 2007, 23, 381–382. (14) Brünger, A. T.; Adams, P. D.; Clore, G. M.; DeLano, W. L.; Gros, P.; Grosse-Kunstleve, R. W.; Jiang, J. S.; Kuszewski, J.; Nilges, M.; Pannu, N. S.; Read, R. J.; Rice, L. M.; Simonson, T.; Warren, G. L. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta. Crystallogr. D. Biol. Crystallogr. 1998, 54, 905–921. (15) Koradi, R.; Billeter, M.; Wüthrich, K. MOLMOL: a program for display and analysis of macromolecular structures. J. Mol. Graph. 1996, 14, 51–55. (16) Laskowski, R. A.; Moss, D. S.; Thornton, J. M. Main-chain bond lengths and bond angles in protein structures. J. Mol. Biol. 1993, 231, 1049–1067. (17) Saitou, N.; Nei, M. The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 1987, 4, 406-425. (18) Tamura, K.; Peterson, D.; Peterson, N.; Stecher, G.; Nei, M.; Kumar, S. MEGA5: Molecular Evolutionary Genetics Analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Mol. Biol. Evol. 2011, 28, 2731-2739. (19) Mygind, P. H.; Fischer, R. L.; Schnorr, K. M.; Hansen, M. T.; Sönksen, C. P.; Ludvigsen, S.; Raventós,

D.; Buskov,

S.; Christensen,

B.; De

Maria,

L.; Taboureau,

O.; Yaver,

D.; Elvig-Jørgensen, S. G.; Sørensen, M. V.; Christensen B. E.; Kjaerulff, S.; Frimodt-Moller, N.; Lehrer, R. I.; Zasloff, M.; Kristensen, H. H. Plectasin is a peptide antibiotic with therapeutic 26


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potential from a saprophytic fungus. Nature. 2005, 437, 975–980. (20) Green, L. C.; Wagner, D. A.; Glogowski, J.; Skipper, P. L.; Wishnok, J. S.; Tannenbaum, S. R. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal. Biochem. 1982, 126, 131-138. (21) Lundberg, J. O.; Weitzberg, E.; Shiva S.; Gladwin M. T. Nitrite and Nitrate in Human Health and Disease, Nutrition and Health. 2011, Part 1, 21-48. (22) Lundberg, J. O.; Weitzberg, E. NO generation from inorganic nitrate and nitrite: Role in physiology, nutrition and the rapeutics. Arch. Pharm. Res. 2009, 32, 1119-1126. (23) Wu, J.; Wang, Y.; Liu, H.; Yang, H.; Ma, D.; Li, J.; Li, D.; Lai, R.; Yu, H. Two immunoregulatory peptides with antioxidant activity from tick salivary glands. J. Biol. Chem. 2010, 285, 16606-16613. (24) Stewart, I.; Schluter, P.; Shaw, G. Cyanobacterial lipopolysaccharides and human health a review. Environ. Health. 2006, 5, 7. (25) Clarke, B. T. The natural history of amphibian skin secretions, their normal functioning and potential medical applications. Biol. Rev. Camb. Philos. Soc. 1997, 72, 365-379. (26) Khandelia, H.; Kaznessis, Y. N. Structure of the antimicrobial beta-hairpin peptide protegrin-1 in a DLPC lipid bilayer investigated by molecular dynamics simulation. Biochim. Biophys. Acta. 2007, 1768, 509-520. (27) Zanetti, M.; Gennaro, R.; Romeo, D. Cathelicidins: a novel protein family with a common proregion and a variable C-terminal antimicrobial domain. FEBS Lett. 1995, 374, 1–5. (28) Ghosh, A. K.; Rukmini, R.; Chattopadhyay, A. Modulation of tryptophan environment in 27



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Page 28 of 41

membrane-bound melittin by negatively charged phospholipids: implications in membrane organization and function. Biochemistry. 1997, 36, 14291-14305. (29) Takeda, K.; Akira, S. TLR signaling pathways. Semin. Immunol. 2004, 16, 3-9. (30) Kawai, T.; Adachi, O.; Ogawa, T.; Takeda, K.; Akira, S. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity. 1999, 11, 115-122. (31) Rosenfeld, Y.; Papo, N.; Shai, Y. Endotoxin (lipopolysaccharide) neutralization by innate immunity host-defense peptides. Peptide properties and plausible modes of action. J. Biol. Chem. 2006, 281, 1636–1643. Table 1. Secondary structural components of cathelicidin-PY in different solutions Solution

Helix(%)a

Beta(%)a

Turn(%)a

Random(%)a

H2O

0.0

49.0

3.1

47.9

1:9

6.4

47.1

3.7

42.7

3:7

21.9

42.6

0.0

35.4

5:5

22.6

41.0

0.0

36.4

7:3

21.5

43.0

0.0

35.4

9:1

21.2

32.5

2.1

44.3

1

49.2

0.0

19.7

31.1

2

26.6

41.4

0.0

32.0

3

27.7

40.5

0.0

31.8

4

29.9

38.2

0.0

31.8

5

25.7

42.1

0.3

32.0

TFE/ H2O (v/v)

SDS (mM)

NaCl (w/v)

28


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0.9%

1.1

52.7

10.3

36.0

1.5

53.3

0.0

45.0

Na2HPO4-NaH2PO4 20mM, pH 7.4 a

Jasco-810 software was used to deconvolute CD spectra into fractional contents and these data

are the average value of three scans.

Table 2. Antimicrobial activity of cathelicidin-PY Microorganisms

MIC (µg/mL)

Gram-negative bacteria E. coli ATCC25922

4.69 (1.37 µM )

E. coli 08A852 (CI, DR)

4.69 (1.37 µM )

E. coli 08A866 (CI, DR)

9.38 (2.74 µM )

P. aeruginosa ATCC27853

18.75 (5.48 µM )

P. aeruginosa 08031205 (CI, DR)

9.38 (2.74 µM )

P. aeruginosa 08031014 (CI, DR)

37.5 (10.95 µM )

Gram-positive bacteria S. aureus ATCC2592

9.38 (2.74 µM )

S. aureus 08A865(CI, DR)

9.38 (2.74 µM )

S. aureus 08A875 (CI, DR)

18.75 (5.48 µM )

B. subtilis ATCC 6633

>100

29



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Fungi C. albicans ATCC2002

4.69 (1.37 µM )

C. albicans 08022821 (CI, DR)

9.38 (2.74 µM )


18.75 (5.48 µM )


4.69 (1.37 µM )

MIC: minimal inhibitory concentration. These concentrations represent mean values of three independent experiments performed in duplicates. CI: clinically isolated strain. DR: drug resistance for ampicillin and benzylpencillin.

Table 3. Antimicrobial activity of cathelicidin-PY in different solutions MIC (µg/mL) Microorganisms Water

NaCl

PBS

CaCl2

MgCl2

E. coli ATCC25922

4.69

2.35

2.35

2.35

2.35

S. aureus ATCC2592

9.38

4.69

4.69

4.69

9.38

P. aeruginosa ATCC27853

18.75

9.38

9.38

18.75

18.75

B. subtilis ATCC 6633

>100

75

>100

75

75

C. albicans ATCC2002

4.69

2.35

2.35

2.35

2.35

MIC: minimal inhibitory concentration. These concentrations represent mean values of three independent experiments performed in duplicates. All the salt concentrations in different solutions are 150 mM. PBS, phosphate buffered solution, Na2HPO4-NaH2PO4, pH 7.4.

Table 4. Bacterial killing kinetics of cathelicidin-PY against E. coli ATCC 25922 Amount of bacteria co-cultured with different samples for different time (CFUa)

30


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Time (min) samples 0

5

10

20

40

60

120

PY×1

45

37.3

23.7

11.3

0

0

0

PY×5

42.7

29.7

10

0

0

0

0

PY×10

46

11.3

0

0

0

0

0

LHSC×1

39.7

45.7

42

31.3

20

11.3

0

LHSC×5

41.7

40.3

29.7

18

8.7

0

0

Control

43.3

47.7

58.3

79.3

131.7

296

1868.3

a

These CFUs represent mean values of three independent experiments performed in duplicates.

PY: cathelicidin-PY. CFU: colony forming unit. LHSC: Levofloxacin Hydrochloride and Sodium Chloride injection. ×1, ×5 and ×10: 1, 5 and 10 times of MIC. The MICs of PY and LHSC against E. coli ATCC25922 are 4.69 and 0.05 µg/mL, respectively.

31



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Figure 1. Purification of cathelicind-PY from the skin secretions of P. yunnanensis. A, Sephadex G-50 gel filtration of skin secretions of P. yunnanensis. The fraction containing antimicrobial activity is marked by an arrow. B, The fraction containing antimicrobial activity from the Sephadex G-50 gel filtration was further purified by C18 RP-HPLC column. The elution was performed with the indicated gradient of acetonitrile at a flow rate of 0.7 ml/min. The purified cathelicidin-PY is indicated by an arrow. C, MALDI-TOF MS analysis of the purified cathelicidin-PY. 194x322mm (150 x 150 DPI)


Page 32 of 41

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Figure 2. The nucleotide sequence encoding cathelicind-PY and the deduced amino acid sequence of the precursor polypeptide. The sequence of mature cathelicidin-PY is boxed. The star (*) indicates the stop codon. 131x108mm (300 x 300 DPI)



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

Figure 3. Evolutionary relationships of cathelicidin-PY and other cathelicidins in vertebrate. The phylogenetic dendrogram is a condensed tree based on Neighbor-Joining and p-distance methods. Only bootstrap values >50% (1000 replicates) are shown next to the branches. Cathelicidin-PY is marked with a triangle (▲). 165x302mm (300 x 300 DPI)


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Figure 4. NMR structure of cathelicidin-PY in in 95% H2O/TFE-d3, 5% D2O. (6:4, v/v, pH 6.2, 25°C), (PDB ID Code: 2LR7). (A) Backbone superimposition of the 20 lowest energy structures. (B) The mean structure calculated from the 20 lowest-energy structures highlighting one disulfide bond (black solid line) and two hydrogen bonds (green broken lines). (C) Ribbon representation of the mean structure of cathelicidin-PY. (D) Electrostatic surface of cathelicidin-PY which takes the same orientation as that in the Panel A. Positively charged region and negatively charged region are shown in blue and red, respectively. The electrostatic surface was calculated and colored using the program MOLMOL. 282x211mm (72 x 72 DPI)



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Figure 5. Effects of cathelicidin-PY on membrane morphology of E. coli by scanning electron microscopy (SEM). A and B, E. coli ATCC 25922. C and D, clinically isolated drug-resistant E. coli 08A852. A and C, control. B and D, treated with cathlicidin-PY (1 × MIC). The breaks of membranes and the flowed outintracellular inclusions are marked by arrows. 248x185mm (300 x 300 DPI)


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Figure 6. Effects of cathelicidin-PY on nitrite and inflammatory cytokines production induced by LPS. A, Nitrite. B, TNF-α . C, IL-6. D,MCP-1. Control, only treated with LPS. Blank, without cathelicidin-PY and LPS. Data are mean ± SEM value of three separate experiments. *P < 0.05, **P < 0.01 significantly different compared to the control. 203x148mm (300 x 300 DPI)



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Figure 7. Effects of cathelicidin-PY on inflammatory response signaling pathways. Raw 264.7 macrophage cells were treated with or without cathelicidin-PY and/or LPS as the indicated concentration. *P < 0.05, **P < 0.01 significantly different compared to the control (100 ng/ml LPS treated only). 249x206mm (300 x 300 DPI)


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Figure 8. Proton spectra of free cathelicidin-PY in presence of membrane-like environment H2O/TFE-d3 (6:4, v/v) (25 °C, pH 6.2, ppm) (A) the TOCSY spectra of cathelicidin-PY. (B) the NOESY connectivity diagram for sequential assignments linked by dαN (the positions of K2 HA -C3 H and N4 HA -F5 H cross peaks are located below the diagonal). 493x705mm (72 x 72 DPI)



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Figure 9. Interaction between cathelicidin-PY and LPS by NMR titration. A, Superposition of the TOCSY spectra of cathelicidin-PY free and in the complex with LPS. The ratio of cathelicidin-PY to LPS was 2.5 mM : 5 µg. B, Column diagram displays the relative peak heights (ratios of the peak heights of amide protons of free peptide to those in the complex with LPS), excluding Asn4, Phe5 (no signals in TOCSY spectra) and Pro27 (without NH) during the titration process. The ratio of cathelicidin-PY to LPS was 2.5 mM : 1 µg and 2.5 mM : 5 µg. 43x47mm (300 x 300 DPI)


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Table of Contents Graphic 57x33mm (300 x 300 DPI)


Structure and Function of a Potent Lipopolysaccharide-Binding

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