Role of Aromatic Amino Acids in Lipopolysaccharide and Membrane Interactions of Antimicrobial Peptides for Use in Plant Disease Control*

KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYT-LR), the representative sequence of helix D of heparin co-factor II, was demonstrated to be potent against agronomically important Gram-negative plant pathogens Xanthomonas vesicatoria and Xanthomonas oryzae, capable of inhibiting disease symptoms in detached tomato leaves. NMR studies in the presence of lipopolysaccharide provided structural insights into the mechanisms underlying this, notably in relationship to outer membrane permeabilization. The three-dimensional solution structure of KYE28 in LPS is characterized by an N-terminal helical segment, an intermediate loop followed by another short helical stretch, and an extended C terminus. The two termini are in close proximity to each other via aromatic packing interactions, whereas the positively charged residues form an exterior polar shell. To further demonstrate the importance of the aromatic residues for this, a mutant peptide KYE28A, with Ala substitutions at Phe11, Phe19, Phe23, and Tyr25 was designed, which showed attenuated antimicrobial activity at high salt concentrations, as well as lower membrane disruption and LPS binding abilities compared with KYE28. In contrast to KYE28, KYE28A adopted an extended helical structure in LPS with extended N and C termini. Aromatic packing interactions were completely lost, although hydrophobic interaction between the side chains of hydrophobic residues were still partly retained, imparting an amphipathic character and explaining its residual antimicrobial activity and LPS binding as observed from ellipsometry and isothermal titration calorimetry. We thus present key structural aspects of KYE28, constituting an aromatic zipper, of potential importance for the development of novel plant protection agents and therapeutic agents.

Recent years have seen a profound increase in the occurrence of multidrug resistant Gram-negative bacterial strains (1,2). Consequently, infections due to these strains pose a considerable, and increasing, threat to human health and economy. This is the case also within agriculture, where large amount of crops are lost every year due to bacterial infections, putting global food security at stake (3). For example, Xanthomonas species are associated with bacterial spot disease in a wide variety of agriculturally important crops, particularly tomato and pepper. They affect leaves, stem, and fruits (4), and are found to be most destructive to crops that are grown in warm and humid weather conditions, leading to a substantial crop loss on a global scale (5). Xanthomonas has been found to display streptomycin resistance since the 1960s (4), thus directing control and prevention toward large scale use of copper-based compounds, mancozeb and/or maneb (6). The latter compounds are carcinogenic as well as toxic to the environment (7). In addition, extensive application of copper compounds has, in itself, led to the development of bacterial resistance in many strains of Xanthomonas (8). Counteracting this, genetic breeding techniques have been successfully applied but are time consuming and suffer from drawbacks that include hybrid weakness and pre-and postzygotic barriers that prevent attainment of fertile hybrids (9,10). Thus, to reduce both environmental and health hazards, new environmentally friendly compounds are needed. Besides, new approaches of plant disease management are also required to meet the current demand of crops.
Within the broader context of multidrug resistance in bacteria, antimicrobial peptides (AMPs) 4 have attracted considerable attention as potential anti-infective drug alternatives (11,12). AMPs are small peptides, Յ50 amino acids, displaying diverse structures as well as biological functions (2,13). They are found in a wide variety of organisms, forming a key part of the innate immunity system, in which they provide antimicrobial properties against a broad range of pathogens (12,14). Most AMPs are cationic and amphipathic in nature, interacting with, and disrupting, negatively charged microbial membranes (2,15,16). Apart from their antimicrobial activities, some AMPs have also been found to display other host defense functions, such as modulation of pro-or anti-inflammatory pathways along with activation of dendritic cells, helping in wound healing and angiogenesis (17,18).
Central for the antimicrobial and anti-inflammatory effects of AMPs against Gram-negative bacteria is their interaction with bacterial lipopolysaccharide (LPS), a key component in the outer leaflet of the outer membrane of Gram-negative bacteria (19 -21). Through electrostatic and steric effects, LPS restricts the entry of AMPs to their site-of-action, i.e. the inner membrane (22)(23)(24). Furthermore, LPS triggers inflammation, causing deregulated expression of proinflammatory cytokines (25). In contrast to AMP interactions with lipid membranes, AMP-LPS interactions remain poorly understood, despite their importance. Further studies on these are therefore needed. In particular, structural elucidations of AMPs in the context of LPS-and membrane-mimicking environments are key for the identification of specific motifs resulting in potent antimicrobial and anti-inflammatory effects against Gram-negative infections.
Earlier studies on heparin co-factor II (HC-II) have demonstrated that helices A and D are responsible for its observed antimicrobial property (26). More recently, the 28-residue peptide KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYTLR) representing the helix D of HC-II ( Fig. 1) has been reported to be highly active against the two Gram-negative bacteria Pseudomonas aeruginosa and Escherichia coli (27). In addition, KYE28 displays immunomodulatory effects, mediating a reduction in proinflammatory cytokines following either endotoxin injection or bacterial infection, although the mechanisms underlying this remain unclear (27).
Taken together, the considerations above initiated our interest in studying LPS binding of KYE28, as well as its influence on devastating Gram-negative plant pathogens belonging to the genus Xanthomonas. To place these investigations in a wider context, results obtained on these plant pathogens were compared with those for more widely investigated clinical pathogens, Klebsiella pneumoniae and Salmonella typhi. Importantly, KYE28 was found to be very active against all tested plant pathogens. In addition to elucidating the membrane disruption underlying this antimicrobial effect through an array of biophysical methods, we also performed NMR studies to elucidate the three-dimensional structure of KYE28 in LPS for better understanding the key residues involved in stabilization of its adopted conformation, and to provide an understanding of the structure-activity correlation. Our studies indicated the importance of the aromatic residues of KYE28 for its interaction with LPS and resulting antimicrobial and anti-inflammatory properties. Therefore, we infer that the aromatic zipper, together with background hydrophobic interactions, is crucial for endowing KYE28 with its bioactivity.
Antimicrobial Activity-To determine the minimal inhibitory concentration (MIC), a microtiter broth dilution method was utilized, as previously described (28). In brief, overnight cultures of the indicated microbes were washed twice and re- suspended in either 10 mM phosphate buffer, pH 7.4, alone or in the presence of 150 mM NaCl, and further diluted to 10 5 cfu/ml. The peptide was dissolved in the same buffer to a stock concentration of 1 mM. 50 l of microbial suspension was added to the corresponding well of a 96-well microtiter plate (polypropylene) together with 50 l of peptide solution to obtain a final peptide concentration ranging from 1 to 100 M. The plate was subsequently incubated at 37°C with rotation for 3 h, after which 200 l of media was added and incubated at the temperature indicated above for 24 to 48 h. The MIC was obtained as the lowest concentration of the peptide where no visual microbial growth was detected. 10 M Polymyxin B was used as a positive control. Microbial suspension in the absence of peptide was used as the negative control.
NF-B Activation Assay-THP1-Xblue TM -CD14 cells (1 ϫ 10 6 /ml) were stimulated with 100 ng/ml of E. coli LPS (Invivo-Gen), with or without the immediate addition of KYE28 or KYE28A. NF-B activation was determined 20 h later by using the Quanti-Blue TM assay (InvivoGen). In brief, through activation of the cells by various antagonists, NF-B/AP-1 activation is induced, leading to the secretion of embryonic alkaline phosphatase into the cell supernatant. For detection, the cell supernatant and a secretion of embryonic alkaline phosphatase detection reagent (QUANTI-Blue TM ) were mixed and absorbance was measured at 600 nm. Results given represent mean values from quadruple measurements.
MTT Assay-Sterile filtered MTT solution (5 mg/ml in PBS) was stored protected from light at Ϫ20°C until use. THP-1 cells from the above experiment were washed, and 20 l of the MTT solution was added to each well and the plates were incubated for 1 h in CO 2 at 37°C. The MTT-containing medium was then removed by aspiration. The blue formazan product generated was dissolved by the addition of 100 l of 100% DMSO per well, and the plates gently swirled for 10 min at room temperature to dissolve the precipitate. The absorbance was monitored at 550 nm, and results given represent the mean values from quandruple measurements.
Circular Dichroidism (CD) Spectroscopy-A Jasco 815 spectrometer was used to study the secondary structures of the peptides in buffer solution as well as in the presence of LPS. Both peptide and LPS stocks were prepared either in 10 mM phosphate buffer, pH 7.4, or in the same buffer with pH 4.5. 25 M peptide was titrated with increasing concentrations of LPS, ranging between 25 and 75 M, and the spectra were recorded at room temperature (25°C) with accumulations of three scans and at a speed of 100 nm/min. Spectra were scanned from 190 to 260 nm, with a data interval of 1 nm. A cuvette having a 0.1-cm path length was used for all measurements. The spectra obtained were corrected using a baseline spectrum in each case. The ellipticity in millidegrees was plotted against wavelength in nm.
NMR Studies-1 mM KYE28/KYE28A solution in 10 mM phosphate buffer, pH 4.5, and 10% D 2 O were used to obtain one-dimensional proton NMR spectra at 25°C, using Bruker Avance III 700 MHz NMR spectrometer equipped with a 5-mm cryoprobe. 4,4-Dimethyl-4-silapentane-5-sulfonate sodium salt (DSS) was used as an internal standard (0.0 ppm) in all NMR experiments. A series of one-dimensional titrations were performed upon addition of increasing concentrations of LPS ranging from 3 to 24 M (E. coli LPS or P. aeruginosa LPS), until line broadening was observed in the proton spectra. Upon significant line broadening (occurring at a LPS:peptide molar ratio of ϳ1:25), two-dimensional homonuclear total correlation spectroscopy (TOCSY) (with mixing time of 80 ms), and transferred nuclear Overhauser effect spectroscopy (trNOESY) experiments with four different mixing times, viz. 75, 100, 150, and 200 ms, were carried out to rule out the possibility of spin diffusion. In addition, trNOESY experiments were carried out for the same sample of the KYE28-LPS complex in 100% D 2 O for unambiguous assignments of aromatic/aliphatic or aromatic/ aromatic NOEs. 24 and 16 scans were performed either for NOESY (peptide in aqueous solution) or trNOESY (LPS-peptide complex with a molar ratio of 1:25) and TOCSY, respectively, per t 1 increment. 16 Dummy scans were performed in each case and a spectral width of 12 ppm was maintained for both experiments along both dimensions. 456 increments in t 1 and 2048 data points in t 2 dimension along with States TPPI (29) for quadrature detection in t 1 dimension and WATER-GATE for water suppression were employed (30). TOCSY and NOESY or trNOESY spectra were processed using 4K (t 2 ) ϫ 1K (t 1 ) data matrices after zero filling. The experiment was processed with Bruker TOPSPIN software and was analyzed using Sparky.
Saturation transfer difference (STD) NMR experiments were conducted with 0.5 mM KYE28/KYE28A dissolved in 100% D 2 O, pH 4.5. LPS stock was also prepared in D 2 O and pH was adjusted to 4.5. A standard STD pulse program (31, 32) using a saturation frequency of Ϫ1 ppm and an off-resonance frequency of 40 ppm yielded the spectra. The experiments were conducted at 25°C. The saturation frequency selectively saturated LPS in solution (on-resonance), whereas the off resonance was such a frequency where neither LPS nor the peptide saw any resonances. An STD experiment was carried out for peptides in the absence and presence of LPS. Total 40 Gaussianshaped pulses (49 ms, 1 ms delay between pulses) with a saturation time of 2 s was employed (33). Subtraction of off-resonance from the on-resonance spectra yielded the saturation transfer difference spectrum (34). 1024 and 512 scans were performed for both STD and reference spectra, respectively, maintaining a spectral width of 12 ppm in each case. A relaxation delay of 2 s was used and 16 dummy scans were carried out for both STD and reference spectra. Before conducting Fourier transformation, an exponential line broadening function of 3 Hz was multiplied with STD one-dimensional spectra.
Structure Calculation-The two-dimensional trNOESY spectra of KYE28 or KYE28A in LPS, recorded at 150 ms mixing time, were used to obtain the upper bound distance constraints from the NOE build up rate, as it contained no spin diffusion. The upper bound distance constraints were calculated with respect to the NOE intensities between the resolved ring protons of either Phe -1 1 (viz. Phe 11 -2H and Phe 11 -3H for KYE28 and ring protons) or Tyr 2 (viz. Tyr 2 -2H and Tyr 2 -3H for KYE28A). The 2.0-Å distance was fixed for lower distance constraints. The calculated distance was then categorized as strong (2.5 Å), medium (2.6 -3.5 Å), and weak (3.6 -5.0 Å) for further structure calculation using CYANA. It is worth mentioning that the folding of the structure depends mainly only on medium-and long-range NOEs. The backbone dihedral angles ⌽ and were varied from Ϫ30 to Ϫ120 and Ϫ120 to ϩ120, respectively, for all nonglycine residues to reduce conformational search. The anti-parallel dimeric structure was also calculated as previously described (35) by connecting two monomer units with a glycine linker consisting of 10 glycines. Sequential, intra-residual, and medium-range NOEs were used to stabilize the monomeric units, whereas long-range NOEs were used as inter-monomeric NOEs. Successive refinements were performed as previously described (36). The 20 lowest energy structures were chosen from the 100 calculated structures to represent the ensemble structure. Procheck (37), PyMOL, and MOLMOL software was used for quality check and visualization of the PDB structures, respectively.
DPH Fluorescence Assay-Fluorescence spectroscopy was used to determine peptide interaction with E. coli LPS. A fluorescence emission spectrum of DPH was obtained in the presence of LPS bicelles, as well as in the presence of increasing peptide concentrations. For the preparation of bicelles, 2 mg of LPS from E. coli 0111:B4 was dissolved in buffer (10 mM phosphate buffer, pH 7.4, 150 mM NaCl) along with 1.51 mg of CHAPSO, keeping q ratio ϭ 0.25. The sample was next subjected to five freeze-thaw cycles and used for further assay.
Bicelle disruption experiments were performed on a Varian Cary Eclipse fluorimeter, using excitation at 358 nm, emission wavelengths of 400 to 600 nm, excitation and emission slit widths of 5 nm, and a photomultiplier detector voltage of 700 V. Measurements were carried out in a 600-l sample volume containing DPH in bicellar buffer (10 mM phosphate buffer, pH 7.4, 150 mM NaCl), which did not show any fluorescence readings. 5 M LPS bicelle was added, which caused a slight fluorescence increase (F 0 ) . To induce the bicelle-disrupting action of the peptide, exposing the hydrophobic acyl group chains of the LPS moieties, the peptide was added in increasing concentrations from 5 M to a final concentration of 35 M. After peptide addition, the increase in fluorescence (F) was monitored after 5 min. The difference, ␦, was calculated as the total fluorescence induced by disruption of the bicelle at a particular concentration of the peptide.
NPN Dye Uptake Assay-Overnight cultures of X. vesicatoria were washed in 10 mM Tris buffer, pH 7.2, containing 150 mM NaCl and re-suspended to a final concentration of 0.5 optical density. 0.5 mM of the fluorescent dye, 1-N-phenylnapthylamine (NPN) was used as the working solution. NPN, from the above working solution, was added to the reaction volume, consisting of 0.5 optical density in 600 l of buffer to a final concentration of 10 M, and allowed to stabilize. Subsequently, increasing concentrations of the peptide were added to the reaction volume, from 5 to 30 M. An increase in fluorescence intensity, on account of the outer membrane permeabilization, was measured using a Varian Cary Eclipse fluorimeter with excitation and emission wavelengths of 350 and 420 nm, respectively. The excitation and emission slit width was 5 nm, whereas the photomultiplier detector voltage used was 700 V.
1% Triton X-100 was used as a positive control, relative to which % of NPN dye uptake was calculated.
Liposome Preparation and Leakage Assay-Model liposomes investigated were anionic (DOPE/DOPG, 75/25, mol/mol), extensively used as a model system in the AMP literature, and previously demonstrated to give similar results regarding peptide interactions as E. coli lipid extract membranes (38). In contrast to the latter, DOPE/DOPG offers methodological advantages. Because of the long, symmetric, and unsaturated acyl chains of these phospholipids, membrane cohesion is good, which facilitates stable, unilamellar, and largely defect-free liposomes and well defined supported lipid bilayers, allowing detailed data on leakage and adsorption density to be obtained. The lipid mixture was dissolved in chloroform, after which solvent was removed by evaporation under vacuum overnight. Subsequently, 10 mM Tris buffer, pH7.4, was added together with 0.1 M carboxyfluorescein (CF) (Sigma). After hydration, the lipid mixture was subjected to eight freeze-thaw cycles, consisting of freezing in liquid nitrogen and heating to 60°C. Unilamellar liposomes of about Ø140 nm were generated by multiple extrusions (30 passages) through polycarbonate filters (pore size 100 nm) mounted in a LipoFast miniextruder (Avestin, Ottawa, Canada) at 22°C. Untrapped CF was removed by two subsequent gel filtrations (Sephadex G-50, GE Healthcare, Uppsala, Sweden) at 22°C, with Tris buffer as eluent. CF release from the liposomes was determined by monitoring the emitted fluorescence at 520 nm from a liposome dispersion (10 M lipid in 10 mM Tris, pH 7.4). An absolute leakage scale was obtained by disrupting the liposomes at the end of each experiment through addition of 0.8 mM Triton X-100 (Sigma). A SPEXfluorolog 1650 0.22-m double spectrometer (SPEX Industries, Edison, NJ) was used for the liposome leakage assay. Measurements were performed in triplicate at 37°C.
Ellipsometry-Peptide adsorption to supported lipid bilayers was studied in situ by null ellipsometry, using an Optrel Multiskop (Optrel, Kleinmachnow, Germany) equipped with a 100milliwatt Nd:YAG laser (JDS Uniphase, Milpitas, CA). All measurements were carried out at 532 nm and an angle of incidence of 67.66°in a 5-ml cuvette under stirring (300 rpm). Both the principles of null ellipsometry and the procedures used have been described in detail before (39). In brief, by monitoring the change in the state of polarization of light reflected at a surface in the absence and presence of an adsorbed layer, the mean refractive index (n) and layer thickness (d) of the adsorbed layer can be obtained. From the thickness and refractive index the adsorbed amount (⌫) was calculated according to, where n 0 is the refractive index of the bulk solution (1.3347), and dn/dc the refractive index increment (0.154 cm 3 /g). Corrections were routinely done for changes in bulk refractive index caused by changes in temperature and excess electrolyte concentration. LPS-coated surfaces were prepared by adsorbing E. coli LPS to methylated silica surfaces (surface potential, obtained from electro-osmosis, Ϫ40 mV, contact angle 90°(40) from 5 mg/ml of LPS stock solution in water at a concentration of 0.4 mg/ml over a period of 2 h. This results in a hydrophobically driven LPS adsorption of 1.48 Ϯ 0.38 mg/m 2 , corresponding to a plateau in the LPS adsorption isotherm under these conditions, with an approximate area per acyl group of 200 Å 2 , assuming an LPS molecular weight of 10 4 , and 6 acyl chains per LPS. Nonadsorbed LPS was removed by rinsing with Tris buffer at 5 ml/min for 30 min, and allowing buffer stabilization for 20 min. The LPS surfaces thus obtained displayed similar peptide adsorption as surfaces for which LPS was incorporated into preformed DOPE/DOPG lipid bilayers (41). Peptide addition was performed at different concentrations of 0.01, 0.1, 0.5, and 1 M, and the adsorption monitored for 1 h after each addition. All measurements were performed in at least duplicate at 25°C.
For lipid A deposition, this was solubilized with 0.25 wt % triethylamine under vigorous vortexing and heating to 60°C for 10 min. Lipid A was adsorbed at methylated silica surfaces for 2 h from 5 mg/ml of lipid A stock solution in 0.25% triethylamine at a concentration of 0.2 mg/ml in 10 mM Tris, pH 7.4, containing 150 mM NaCl. Non-adsorbed lipid A was subsequently removed by rinsing with the same buffer at 5 ml/min for 15 min, followed by buffer stabilization for 20 min. This results in a lipid A adsorption of 0.8 Ϯ 0.2 mg/m 2 . Peptide addition was subsequently performed to 0.01, 0.1, 0.5, and 1 M, and adsorption was monitored for 1 h after each addition. (Absence of transient maxima in the time-resolved adsorption curves after peptide addition indicated competitive displacement to be unlikely for the LPS and lipid A substrates.) All measurements were performed in at least duplicate at 25°C.
Supported lipid bilayers were generated by liposome adsorption. Anionic DOPE/DOPG (75/25 mol/mol) liposomes were prepared as described above, but the dried lipid films were resuspended in Tris buffer only with no CF present. To avoid adsorption of peptide directly at the silica substrate (surface potential Ϫ40 mV, contact angle Ͻ10°) (39) through any defects of the supported lipid layer, poly-L-lysine (molecular mass ϭ 170 kDa, Sigma) was preadsorbed from water prior to lipid addition to an amount of 0.045 Ϯ 0.01 mg/m 2 , followed by removal of nonadsorbed poly-L-lysine by rinsing with water at 5 ml/min for 20 min (42). Water in the cuvette was then replaced by buffer also containing 150 mM NaCl, followed by addition of liposomes in buffer at a lipid concentration of 20 M, and subsequently by rinsing with buffer (5 ml/min for 15 min) when the liposome adsorption had stabilized. The final layer formed had structural characteristics (thickness 4 Ϯ 1 nm, mean refractive index 1.47 Ϯ 0.03), suggesting that a layer fairly close to a complete bilayer is formed. After lipid bilayer formation, the cuvette content was replaced by 10 mM Tris buffer at a rate of 5 ml/min over a period of 30 min. After stabilization for 40 min, peptide was added to a concentration of 0.01 M, followed by three subsequent peptide additions to 0.1, 0.5, and 1 M, in all cases monitoring the adsorption for 1 h. All measurements were made in at least duplicate at 25°C.
Isothermal Titration Calorimetry (ITC)-A VP-ITC microcalorimeter (Micro Cal Inc., Northampton, MA) was used to perform the ITC experiments in triplicates to assess the binding interaction of KYE28/KYE28A with LPS. Peptide and lipid stocks were prepared in 10 mM phosphate buffer, pH 7.4, and degassed. A sample volume of 300 l of 50 M LPS was titrated with KYE28/KYE28A at rate of 2 l/injection at an interval of 180 s, at 308 K, in a set of 20 injections. The raw data were plotted using MicroCal Origin 5 software to obtain thermodynamic parameters, including the association constant (K a ), change in enthalpy (⌬H), free energy of binding (⌬G), and entropy (⌬S). The data were fitted in a single site binding model using Equations 3 and 4.
Detached Leaf Assay-Healthy leaves from 60-day-old tomato plants were excised with a sterile blade and placed on wet tissue in separate sterile Petri plates. The leaves were grouped into three, representing control, infected (bacteria alone), and treated (bacteria ϩ peptide) sets. A log phase X. vesicatoria culture, washed twice and re-suspended in 10 mM phosphate buffer corresponding to 4 optical density was used to set up the reaction. 50 l of bacterial suspension was incubated with 50 l of peptide at 50 M concentration. A control was maintained containing 50 l of bacterial suspension and 50 l of buffer. All reactions were incubated at 28°C for 3 h. 10 l of the reaction mixture was loaded on the lower surface of each leaf and spread all over the surface. Autoclaved buffer was applied to the control set of leaves. Only bacterial suspension was applied to the infected set. The peptide-treated bacterial suspension was applied to the treated set of leaves. The leaves were kept at 25°C in a photoperiod of 12 h of light and 12 h of dark. The leaves were observed for symptom development until 10 to 12 days post-infection. The experiment was conducted in triplicates. Three biological replicates were used.

Results and Discussion
Solution Structure of KYE28 in LPS Micelle-Recent investigations on KYE28 had shown potent activity of the peptide against Gram-negative E. coli and P. aeruginosa. It demonstrated a reduction in the levels of LPS-induced pro-inflammatory cytokines both in vitro and in mouse models. In addition, the peptide was able to neutralize LPS itself very effectively. To analyze the structural parameters responsible for these activities, we here carried out a structural characterization of KYE28 in LPS. First, we investigated the secondary structure of the peptide in the presence of LPS using CD spectroscopy. The CD spectrum of KYE28 in aqueous solution showed a strong negative band near 200 nm corresponding to random coil conformation of the peptide in free state at both pH 7.4 (physiological pH) as well as at pH 4.5 (NMR experimental condition). This is in accordance with previous reports that suggest that many AMPs assume random coil conformations in aqueous solution (41). Upon addition of LPS at a concentration greater than its CMC, a drastic change occurred in the CD profile at both pH conditions (pH 4.5 and 7.4), indicating structural changes in KYE28 upon binding to LPS (Figs. 2A, left panel, and supplemental S1, upper panel). A positive maxima at 195 nm and two strong negative peaks, one at 208 nm and a shoulder of lower intensity at 222 nm, were observed, suggesting an ␣-helical conformation of the peptide in LPS under both pH condi- tions. Deconvolution of the CD spectrum using CDNN software (43) showed a helical content of ϳ20%, turn conformation of 18%, parallel and antiparallel ␤ sheet conformation of 10 and 12%, respectively, and random coil conformation of ϳ40% ( Fig.  2A, right panel). However, it may be noted that deconvolution with CDNN is merely supportive and only included for comparison purposes, considering that such evaluations may be skewed to some extent by the databases being based on globular proteins rather than linear amphipathic peptides. To gain further insight into the KYE28 structure in LPS at the atomic resolution, small amounts of LPS micelles were added to a sample containing KYE28 and monitored using one-dimensional 1 H NMR spectra (44). In doing so, line broadening was observed for peptide signals (without causing any chemical shift perturbation), indicating fast conformational exchange between free and bound forms of the peptide. It is important to note here that LPS forms high molecular weight aggregates/micelles at very low concentrations (14 g/ml) (45). This motivated us to determine the three-dimensional structure of KYE28 bound to LPS micelles with the well known trNOESY method used to study macromolecule-ligand interaction having K d values in the millimolar to micromolar range (22,46). The two-dimensional 1 H-1 H NOESY spectra of KYE28 in aqueous solution showed only intra-residual and sequential NOEs between the backbone and side chain protons (supplemental Fig. S2), supporting lack of any folded conformation, as obtained from CD spectrum as well. In contrast, the trNOESY spectrum of KYE28 in the presence of LPS was characterized by a large number of mediumand long-range NOE cross-peaks ( Fig. 2B and supplemental  Fig. S2). Additionally, the trNOESY spectra of KYE28 in both E. coli and P. aeruginosa LPS showed a perfect superimposition, indicating that the peptide adopted a similar structure in both contexts, irrespective of different O-antigen moieties (Fig. 2C). As seen in Fig. 2, B and D, medium-range NOEs ␣N (i, iϩ2/ iϩ3/iϩ4) were seen between residues Tyr 2 /Thr 6 , Ile 4 /Ile 7 , Ile 4 / His 8 , Thr 6 /His 8 , His 8 /Leu 10 , His 16 /Phe 19 , Asn 22 /Gly 24 , and Phe 23 /Tyr 25 . In addition, there were several side chain NOEs between aliphatic amino acid residues and aromatic ring protons (Fig. 2B). There were also a large number of long-range unambiguously assigned trNOE contacts between Thr 6 /Tyr 25 , Leu 10 /Phe 19 (Fig. 2B, supplemental Table  S1). Note that the aromatic peaks for Phe residues 11, 19, and 23 of KYE28 were unambiguous in this sample condition. A closer look at the fingerprint regions of the spectra revealed several ␣N (i,iϩ2/iϩ3/iϩ4) trNOEs for the residues from Lys 1 to Phe 11 and from His 16 to Gly 24 , signifying ␣ helical pattern in these regions. Also, a plot of ⌬H ␣ (deviation in chemical shift values) of the C ␣ H against the amino acid residues showed consecutive upfield shifted values from the chemical shifts of random coil conformation, for residues Lys 1 to Phe 11 and His 16 to Gly 24 , confirming ␣ helical populations in these segments (supplemental Fig. S3). In addition, NN(i,iϩ2) trNOEs between Ile 7 / Asn 9 , Asn 9 /Phe 11 , and Phe 19 /Arg 21 (Fig. 2, B and D) were also observed. Collectively, aromatic amino acid residues (Tyr 2 , Phe 11 , Phe 19 , Phe 23 , and Tyr 25 ) and hydrophobic amino acid residues (Ile 7 , Leu 10 , and Leu 27 ) of KYE28 showed major hydrophobic interactions (Fig. 2E).
The three-dimensional structure of KYE28 in LPS was calculated using the NOE distance constraints obtained from the trNOESY spectra. Long-range NOE contacts between residues located at the two termini, notably Tyr 2 /Tyr 25 , Thr 6 /Tyr 25 , Tyr 2 /Thr 26 , and Tyr 2 /Leu 27 , suggested the possibility of formation of an anti-parallel dimer of KYE28. Therefore, we determined an antiparallel dimer conformation of KYE28 using the above short-, medium-, and long-range NOEs. However, the backbone and side chain root mean square deviation values of the calculated anti-parallel dimer conformation clearly indicated the lack of a well defined structure, as also evident from the poor superimposition of the backbone and side chain of 20 ensemble structures (supplemental Fig. S4, A and B). Only some of the long-range NOE contacts, such as Leu 10 /Phe 19 and Phe 11 /Phe 19 , were satisfied in the antiparallel dimer conformation when used as inter-monomeric contacts (supplemental Fig. S4 C, marked in blue). In contrary, the majority of the prominent as well as unambiguously assigned long-range NOEs such as Tyr 2 /Tyr 25 , Thr 6 /Tyr 25 , Leu 18 /Phe 23 , Phe 11 /Tyr 25 , and Phe 19 /Tyr 25 (supplemental Fig. S4C, marked in red) were incompatible and were not satisfied in the anti-parallel dimer conformation (supplemental Fig. S4B) due to the large violations in distance constraints (Ͼ1 Å) upon structure calculation. Consequently, no NOE cross-peaks were observed, correspondingtotheshortinter-protondistancesbetweentwomonomeric units of the anti-parallel dimer conformation, viz. C ␣ H/ C ␣ H or NH/NH NOE contacts (Fig. 2B). A parallel dimeric structure was similarly not feasible using the set of observed NOEs in the spectra.
Considering the incompatibility of the experimental results with an anti-parallel dimeric structure, we calculated a monomeric structure using the obtained NOE constraints. Interestingly, it gave rise to a structure closely resembling a "helix-loophelix" motif, stabilized by an aromatic zipper (Fig. 3B) that showed a good convergence of backbone atoms (C ␣ , N, and CЈ) of all the 20 lowest energy structures (Fig. 3A), with an average backbone and heavy atom root mean square deviation values of 0.66 and 1.46 Å, respectively ( Table 1). The LPS-bound folded structure of KYE28 was characterized by an N-terminal helical region Lys 1 -Phe 11 , followed by a loop, a short helical stretch, and an extended C terminus (Fig. 3B). This is in agreement with the deconvoluted CD data denoting the percentage secondary structure of KYE28 in LPS. The structure was stabilized by aromatic packing interactions majorly between Tyr 2 /Tyr 25 (Fig.  3B). This also correlates well with recent findings of a partial activity loss for KYE21 and NLF20, fragments obtained from KYE28 lacking the C-terminal and N-terminal ends, respectively (41). It is interesting to note that the positively charged Arg and Lys residues remain flexible and oriented at one particular face (Fig. 3B, highlighted), forming a polar exterior shell that may interact with the negatively charged phosphate and carboxyl groups of LPS. All aliphatic and aromatic residues, in turn, were packed together forming a core that may help the peptide to penetrate into the lipid bilayer. The electrostatic potential map also supports this observation (Fig. 3C).
The aromatic-aromatic or aromatic-aliphatic packing, which resulted in bringing the two terminal ends in close proximity to each other, as determined using trNOESY methods in the presence of LPS, is common for many AMPs. One such example is the recently reported 16-residue AMP, designed from the dengue viral fusion peptide, which also had a single N-terminal helical turn and a looped out structure, with N and C termini brought close to each other via packing interactions between the centrally located Trp 5 and Phe 12 (28). Similarly, MSI-594 (36), consisting of 24 amino acid residues (PDB acquisition code 2K98), or paradaxin (22), consisting of 33 amino acid residues (PDB acquisition code 2KNS), in LPS also adopt helical hairpin conformations to stabilize the peptide through Phe and other aliphatic amino residues. Therefore, AMPs with helical hairpin or helix-loop-helix motifs, stabilized via aromatic-aliphatic residue interactions seem to be favorable for permeabilization of the outer membrane of Gram-negative bacteria. Furthermore, helical conformations have been previously suggested to stabilize folded structures of AMPs on the bacterial membrane surface, helping in anchoring, which subsequently leads to their translocation and membrane disruption (47,48). Based on this, we propose that the N-terminal helical portion of KYE28 helps in anchoring the lipid membrane, translocating the peptide into the outer leaflet and stabilizing it through hydrophobic interactions between the acyl chains of LPS and the peptide hydrophobic core.
Design and Antimicrobial Activities of the Mutant Peptide KYE28A-To further probe the importance of aromatic packing interactions involved in the antimicrobial activity of KYE28, we carried out Ala substitutions at the critical aromatic residues Phe 11 , Phe 19 , Phe 23 , and Tyr 25 , which were primarily seen to be involved in stabilizing the folded conformation of KYE28 bound to LPS. This resulted in the analogue KYE28A (Fig. 1). Next, to monitor the consequences of these substitutions, antimicrobial assay of both the parent peptide KYE28 and the mutant KYE28A were performed against Gram-negative plant pathogens X. oryzae and X. vesicatoria in addition to S. typhi, K. pneumoniae, and the fungal pathogens C. albicans and C. neoformans. It should here be noted that X. oryzae and   (Table 2). KYE28 Exerts Antimicrobial Activity through Membrane Disruption-Further biophysical studies were performed with both the native and mutant peptide to assess the membrane destabilizing behavior of the peptides. An increased DPH fluorescence in the presence of LPS bicelles, or in the presence of E. coli total lipid extract bicelles, upon peptide addition was taken as a measure of bicelle disruption mediated by peptide anchoring and translocation. In aqueous solution, DPH showed only weak fluorescence, which, however, is enhanced by peptide-induced disruption of bicelles and exposure of the hydrophobic acyl chains of the lipids to DPH. The native peptide KYE28 was found to cause disruption of both E. coli total lipid extract bicelles (Fig. 4A, left panel) and LPS bicelles (Fig. 4A,  right panel) in a concentration-dependent manner, evident from a steady increase in the DPH fluorescence intensity. In contrast, KYE28A caused disruption of the bicelles to a much lower extent (Fig. 4A), showing an ϳ6-fold lower increase in DPH intensity in the case of E. coli total lipid extract bicelle and ϳ12-fold lower increase in the case of LPS bicelle. It may be noted that KYE28 showed a greater increase in DPH intensity in LPS bicelles, starting from 20 M and reaching 2-fold higher values at 35 M peptide concentration when compared with E. coli total lipid extract bicelles. This may be attributed to either a preferential binding of KYE28 to LPS bicelles causing increased disruption, or effects of the difference in the hydrophobic environments displayed by LPS and the E. coli total lipid extract component lipids with which DPH interacts upon bicelle disruption. KYE28A, however, showed similar patterns of fluorescence increase in both cases. Next, outer membrane permeabilization of X. vesicatoria was determined using NPN dye uptake assay. NPN, being a neutral hydrophobic fluores-cent probe, generally remains excluded by the outer membrane of Gram-negative bacteria. However, the dye exhibits an increase in fluorescence intensity on interaction with, and disruption of, the parted outer membrane. A steady increase in NPN fluorescence was observed in a concentration-dependent manner, showing a maximum of around 80% increase in NPN uptake upon addition of 30 M KYE28 peptide (Fig. 4B), whereas a 66% increase in NPN uptake was observed for as low as 1 M KYE28 (i.e. its MIC) (Fig. 4B). In contrast, KYE28A showed only 40% increase in NPN uptake at a peptide concentration of 30 M (Fig. 4B). Taken together, these data further support the importance of the aromatic residues in KYE28 that upon Ala substitution resulted in attenuated activity. In addition, carboxyfluorescein leakage from DOPE/DOPG (75/25 mol/mol) vesicles as a consequence of peptide binding and vesicle disruption was studied.KYE28andKYE28Abothdisplayedconcentration-dependent permeabilization of DOPE/DOPG liposomes (Fig. 4C). Quantitatively, KYE28 was found to be only slightly more potent than KYE28A in causing membrane rupture, demonstrating that under such low ionic strength conditions, electrostatics dominate as the driving force for lipid membrane de-stabilization as both KYE28 and KYE28A have the same number of positively charged Arg and Lys residues in place. This is in agreement with the antimicrobial activity assay data, where KYE28A displayed some antimicrobial effect in the absence of salt, but failed to do so at the same concentrations in the presence of salt.
Binding Interactions of KYE28 and KYE28A to Lipid Bilayers-To elucidate binding interactions between KYE28/ KYE28A and LPS or DOPE/DOPG bilayers, ellipsometry measurements were carried out. In analogy to anionic DOPE/ DOPG membranes, both KYE28 and KYE28A display extensive binding to E. coli LPS, as well as to its lipid A moiety (Fig. 5, A  and B). This is reasonable, because both peptides are strongly positively charged, and KYE28 also quite hydrophobic, whereas LPS carries a negative charge due to its phosphate and carboxylate groups (49) and its lipid A moiety is obviously also hydrophobic. In analogy to other hydrophobically modified biopolymers, e.g. peptidoglycans (50), LPS is likely to interact with the hydrophobic ( Ϸ 90 o ) and negatively charged (z ϷϪ40 mV) methylated silica surface through its lipid A moiety. At the relatively high LPS densities reached in these experiments (1.4 mg/m 2 ), the lipid A moiety will therefore be partly screened by the LPS carbohydrate chains, hence not fully accessible to peptide binding. Because the carbohydrate moieties are much longer than lipid A, they are expected to have a larger peptide binding capacity than lipid A (51). Despite this, KYE28 binding is quite comparable for LPS and lipid A, suggesting a higher binding affinity of this peptide to lipid A. KYE28A, on the other hand, displays a considerably lower binding affinity for lipid A, an obvious consequence of the deletion of aromatic amino acids in this peptide. As can be seen in Fig. 5C, both KYE28 and KYE28A display concentration-dependent binding to DOPE/ DOPG bilayers, reaching adsorption densities at 1 M of 330 and 276 nmol/m 2 , corresponding to about 10 and 12 lipid molecules per peptide, respectively. The lower membrane binding displayed by KYE28A is due to the absence of the aromatic residues, in line with previous findings of the membrane-binding driving force provided by such aromatic amino acid residues (52). ITC experiments corroborated this. Downward profiles of ITC suggest an exothermic or enthalpy driven reaction in both the cases of KYE28 (Fig. 5D) and KYE28A (Fig. 5E) binding to LPS. Quantitatively, however, the binding interaction of KYE28 to LPS was found to be substantially stronger, having a dissociation constant of 35 M, a ϳ5 fold lower value compared with that of KYE28A. Again, this signifies a stronger binding of KYE28 to LPS because of the aromatic residues Tyr 2 , Phe 11 , Phe 19 , Phe 23 , and Tyr 25 that were crucial in mediating hydrophobic interactions with the acyl chains of LPS. All the thermodynamic parameters of KYE28/KYE28A binding to LPS are summarized in Fig. 5, D and E. Structural Aspects of KYE28A in LPS-To elucidate the consequences of alanine mutations on the structural aspects of KYE28A, CD experiments were performed in the presence of LPS. The CD spectrum of KYE28A alone in aqueous solution was characteristic of a random coil nature. However, upon addition of LPS, a positive maxima at 195 nm and two negative maxima at 208 and 222 nm appeared, a signature of ␣ helix conformation. Surprisingly, the molar ellipticity peak at 222 nm was lower in intensity and broader in nature, indicating a large extent of dynamicity in the ␣-helical content (Fig. 6A). CD data deconvolution reflected an ␣-helical content, amounting to 28% of the total. In addition, 37.5% random coil conformation, FIGURE 4. Membrane permeabilization effects of KYE28 and KYE28A. A, bar plots showing the increase in DPH fluorescence as a function of peptide concentration upon addition of peptide to E. coli total lipid extract bicelle (left) and LPS bicelle (right) as a measure of bicelle disruption. KYE28 is seen to cause a pronounced concentration-dependent increase in DPH fluorescence in both E. coli total lipid extract bicelles and LPS bicelles, much larger than those caused by KYE28A. B, plot showing percentage of NPN dye uptake by X. vesicatoria cells upon addition of KYE28 and KYE28A. More than 2-fold higher uptake is seen for KYE28 as compared with KYE28A. C, percentage of dye leakage from DOPE/DOPG (75/25 mol/mol) vesicles as a function of peptide concentration. Both KYE28 and KYE28A induced comparable dye leakage from vesicles, KYE28 producing slightly greater effects.
15.5% turn conformation, and nearly 10 and 9% in parallel and antiparallel ␤ sheet conformation, respectively, were observed for KYE28A in LPS (Fig. 6A). To correlate the structural attributes of KYE28A with its observed attenuation in antimicrobial activity in the presence of salt and retention of only partial activity in the absence of salt, we elucidated its three-dimensional structure, using a two-dimensional 1 H-1 H trNOESY experiment in the presence of LPS. The trNOESY spectra revealed only sequential ␣N/NN(i,iϩ1) and medium-range ␣N(i,iϩ2/iϩ3) NOE contacts (Fig. 6, B and D). No long-range NOEs were observed (Fig. 6E). The superposition of aromatic regions of the trNOESY spectra of KYE28 (marked in red) and KYE28A (marked in blue) in the presence of LPS (Fig. 6C) thus indicated that there were no long-range NOE contacts of KYE28A in the presence of LPS. ␣N(i,iϩ3/iϩ4) NOE contacts were seen between residues Ile 4 /Ile 7 , Thr 6 /Asn 9 , Lys 13 /His 16 , Lys 13 /Arg 17 , and between Leu 14 /Arg 17 or Leu 14 /Leu 18 (Fig. 6B), which is a signature of ␣-helix formation. An ␣N(i,iϩ2) NOE contact was also seen between Ile 4 and Thr 6 (Fig. 6B). The ⌬H␣ values of C ␣ H were furthermore, seen to be shifted upfield in LPS for residues Tyr 2 to His 8 and Leu 14 to Phe 19 (supplemental Fig. S3), signifying that these regions were ␣-helical in nature.
However, smaller deviations in the chemical shift values for KYE28A compared with KYE28 indicated that the KYE28A structure was a loosely bound one. This data agrees well with the CD results (Fig. 6A). As a consequence, KYE28A showed fewer NOE cross-peaks in LPS (Fig. 6E) compared with KYE28 in LPS (Fig. 2E). This indicated the absence of a well defined structure of KYE28A in the presence of LPS, as also evident from an absence of convergence of the 20 lowest energy structures (Fig. 6F), as well as high root mean square deviation values for both backbone and heavy side chain atoms (Table 1). With these reservations, the three-dimensional structure of KYE28A (Fig. 6G) showed an N-terminal helical segment from residues Tyr 2 to Leu 18 and an extended C terminus, where the ␣-helix discontinuation at Ala 19 could be due to the presence of the two consecutive positively charged Arg residues of Arg 20 and Arg 21 . This is supported by reports on a centrosymmetric designed ␣-helical peptide motif of 7 residues according to which a hydrophobic residue at the 12th position and a positively charged residue at the 9th and 13th position would favor the formation of a continuous helix (53). The presence of an Asn residue, Asn 22 , may also contribute to the break as Asn is usually found in turns or in turns of ␤ sheets due to the fact that its side chain can form hydrogen bond with the peptide backbone, thereby disrupting the ␣-helical structure (54). The peptide structure revealed that the positively charged residues Arg 12 , Lys 13 , Arg 17 , and Arg 20 were present at the central region of KYE28A and oriented toward one face of the helix, maintaining a specific pattern dictated by a motif 12 RXXXH 16 XXXR 20 and 13 KXXXR 17 XXXR 21 (Fig. 6G). This motif is thought to be primarily responsible for governing electrostatic interactions with negatively charged phosphate head groups of LPS (23). It is furthermore, interesting to note the orientation of His 16 , which allowed it to be placed in the polar face of the molecule, aiding it to interact with the negatively charged phosphate head group of LPS. Overall, stabilization of the KYE28A structure in the presence of LPS is predominantly electrostatically driven, due to the presence of 8 positively charged amino acid residues in its sequence. In contrast, the absence of aromatic residues in KYE28A led to a destabilization of its hydrophobic core and formation of a longer helical segment, mediated by the substi-tuted alanine. It is to be noted that alanine is well known to have a high ␣-helical propensity among all amino acids (53,55). Apart from the strong electrostatic interaction between positively charged amino acid residues of KYE28A and phosphate head groups of LPS, the peptide was also stabilized by hydrophobic interactions such as 4 IXXI 7 XXXA 11 XXL 14 XXXL 18 in the hydrophobic face of the structure. This observation of binding of either KYE28 or KYE28A to LPS is well supported by ellip-  Overall, this study provides molecular insights into the mechanism of action of KYE28. An aromatic zipper was found to be principally responsible for its biological activities, whose substitution with alanine in KYE28A led to an amphipathic but bent helical structure (Fig. 7A) with much reduced activity both in terms of antimicrobial effects in presence of high salt concentrations and LPS binding and neutralization. Again, this is analogous to findings on a mutant of a designed peptide, MSI-594, for which a Phe 5 was substituted with an Ala, leading to disruption of the helix-loop-helix motif and formation of a curved helix (Fig. 7B) (36). Thus, it was found that the Phe 5 residue in MSI-594 plays a crucial role in inter-helical packing interactions, removal of which completely abolished its broad spectrum antimicrobial activity against Gram-negative bacteria and led to reduced binding affinity to LPS. Similarly, in Pardaxin, a naturally occurring antimicrobial peptide found in sole fishes, Phe 15 was reported to play a major role in mediating hydrophobic contacts between the nonpolar faces of the two helical segments (24) (Fig. 7C).
Residue-specific Interactions of KYE28/KYE28A with LPS-Residue-specific interactions of KYE28 and KYE28A with LPS were probed using STD NMR spectroscopy. This method is well known to recognize residues or groups in a ligand molecule that are involved in direct interaction with their high molecular weight binding partners. Fig. 8 shows the reference one-dimensional proton spectrum of KYE28 (Fig. 8A) and KYE28A (Fig.  8B) and their corresponding STD spectra. In case of KYE28, large STD effects were observed for the aromatic proton resonances resonating between 6.7 to 7.5 ppm due to the ring protons of Tyr 2 /His 8 /Phe 11 /His 16 /Phe 19 /Phe 23 /Tyr 25 . Among these STD effects, unambiguous assignments were possible for Phe (Phe 11 , Phe 19 , and Phe 23 ) aromatic protons of KYE28. This signifies the close proximity of these residues to LPS micelles. In addition, strong STD effects were observed between 0.8 and 1.3 ppm, corresponding to the alkyl side chain groups of Ile/Leu residues. Moderate STD effects were also observed between 1.4 and 1.9 ppm, which could originate from the alkyl side chain groups of the positively charged Lys/Arg residues. Moderate to low STD effects were also observed for the ␤ protons of the aromatic residues resonating between 2.8 and 3.3 ppm. This provides additional evidence for the involvement of the aromatic residues in stabilization of the bound conformation of KYE28 in LPS micelles through hydrophobic interactions. In contrast, the STD spectrum of KYE28A quite obviously lacked most of the STD effects from the aromatic resonances. Instead, weak STD effects were observed for the ring proton and ␤ protons of Tyr 2 . Strong STD effects were observed between 0.8 and 1.3 ppm, corresponding to the alkyl side chain groups of Ile/Leu and the substituted Ala, showing their involvement in the interaction and stabilization of KYE28A in LPS micelles. Moderate STD effects, corresponding to the side chain groups of the positively charged residues, similar to those of KYE28, were also observed between 1.4 and 1.9 ppm. Due to severe signal overlap in certain regions of the one-dimensional STD spectra in both cases, however, residue-specific information could not be obtained for all participating residues of KYE28/KYE28A. Nevertheless, total STD effects from the methyl protons of Leu/Ile (resonating between 0.8 and 1 ppm) and ␤/␥ protons of Leu/Ile/Lys/Arg (resonating between 1 and 1.8 ppm) in the case of KYE28 were found to be 2-fold higher than for KYE28A, signifying that these residues in KYE28 were more closely placed in the LPS micellar environment. Taken together, these studies reveal the importance of the aromatic, hydrophobic, and positively charged residues in stabilization of the bound form of KYE28 in LPS.
Anti-inflammatory Effects of KYE28 and KYE28A-To assess to what extent the differences in LPS binding between KYE28 and KYE28A influence the anti-inflammatory effects of these peptides, the ability of the peptides to block LPS-triggered NF-B activation in macrophages was investigated. As shown in Fig. 9, KYE28 suppressed NF-B activation in a dose-dependent manner, with sizeable suppression already at 0.5-1 M, i.e. around the MIC of this peptide. In contrast, KYE28A displayed essentially no blocking of NF-B up to a peptide concentration of 50 M. As shown by the corresponding MTT assay results, these effects are indeed due to differences in peptide blocking of   LPS activation, and not due to peptide toxicity effects. Thus, the differences observed regarding peptide binding amount/affinity from ellipsometry and ITC, and regarding conformational aspects observed by the different NMR methodologies, do indeed translate to the anti-inflammatory function of these peptides. Conversely, this correlation between biological function and peptide structure on LPS binding suggests that the structural zipper motif identified may be a promising feature to aim for in the development of novel antimicrobial and anti-inflammatory peptide therapeutics and plant protection peptides.
Ex Vivo Inhibition of X. vesicatoria Infection by KYE28 -The low MIC of KYE28 against the plant pathogen X. vesicatoria in the presence of high salt concentrations motivated us to study its effects in inhibiting disease in plants to obtain initial information on the feasibility of its use in treating plant diseases. Inhibition of X. vesicatoria disease symptoms in detached tomato leaves, as obtained from the detached leaf assay, revealed that upon treatment with KYE28 at a concentration of 50 M, the disease symptoms were not seen in the treated set of leaves. This is in stark contrast to the infected leaves that had visible black spots on the leaf surface, a characteristic of X. vesicatoria infection (Fig. 10). Potent activities of the peptide against X. vesicatoria even in the presence of high salt concentrations may have important biological implications in application of the peptides to alleviate symptoms in diseased plants. Therefore, inhibition of X. vesicatoria disease in detached tomato leaves is encouraging enough to study the effects of the peptide in treating plant disease through external application or genetic engineering. Thus, there is further scope in analyzing the efficacy of the peptide in treating plant disease in vivo (development of transgenic line over-expressing the KYE28 peptide).
In conclusion, rationally designed peptides provide a promising approach toward development of novel therapeutics endowed with improved antimicrobial and anti-inflammatory properties (56 -58). In this broader context, the structural insights provided by KYE28 in LPS, identifying an aromatic zipper motif playing a crucial role in endowing KYE28 with potent antimicrobial activity, may help in the development of potent new antimicrobials with enhanced biological properties.