Structural Characterization of Antimicrobial Peptides from Lactobacillus salivarius K4 by Circular Dichroism ABSTRACT

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1 Structural Characterization of Antimicrobial Peptides from Lactobacillus salivarius K4 by Circular Dichroism Kannika Thongkhao 1, Anchanee Kubera 3, Sunee Nitisinprasert 4 and Kiattawee Choowongkomon 1,2,* ABSTRACT Salvicin K (Sal K) and antimicrobial-like bacteriocin β (Alb β), which are cationic antimicrobial peptides (AMPs) from Lactobacillus salivarius K4, have been reported as novel bacteriocins from lactic acid bacteria. They are heat stable, and are able to inhibit some foodspoilage pathogens, especially the gram-positive bacteria. However, their structural information and mode of action are still unknown. Hence, in this study, we aim to characterize their secondary structures by using circular dichroism (CD).The obtained structures will be used for further studies. The experiments were performed in various buffers, which were unbuffered water, 2 mm DPC, ph 4.0 and 10% SDS ph 4.0, at 25 C. In unbuffered water condition, the Sal K and Alb β alone were unstructured while their mixture adopted partial structure of unstructured and α-helix. This result indicated that Sal K and Alb β interacted which affected their secondary structures. In 2 mm DPC micelles, ph 4.0, the Sal K peptide adopted helical conformation. Nevertheless, in 10% SDS, ph 4.0, Sal K and Alb β alone and the peptide mixture adopted helical structure with and 94.11, respectively of α-helix, indicating that these peptides interacted with SDS micelles and adopted helical structure. However, further experiments need to be performed to gain more detail between the peptide-peptide interaction, peptide-micelles binding and their modes of action.. Key words: Structural characterization, antimicrobial peptide, Lactobacillus salivarius K4, Circular dichroism *Corresponding author; address: *fsciktc@ku.ac.th 1 Interdisciplinary Graduate Program in Genetic Engineering, Graduate School, Kasetsart University, Bangkok, 10900, Thailand 2 Department of Biochemistry, Faculty of Science, Kasetsart University, Bangkok, 10900, Thailand 3 Department of Genetics, Faculty of Science, Kasetsart University, Bangkok, Thailand 4 Department of Agro-Industrial Technology, Faculty of Agro-Industry, Kasetsart University, Bangkok, Thailand

2 INTRODUCTION Salvicin K (Sal K) and antimicrobial-like bacteriocin β (Alb β) are antimicrobial peptides (AMPs) or bacteriocins from Lactobacillus salivarius K4. They were first identified, isolated and characterized from lactic acid bacteria in chicken intestine (Pilasombut, 2006). Sal K has been purposed as a novel antimicrobial peptide because it is heat tolerant up to 100 C for 5 min and it antimicrobial activity decreased by 50% after extended heating at 100 C for 30 min, no amino acid sequence homology and against food pathogens such as Listeria innocua and Bacillus coagulans (Pilasombut, 2006). Recently, Sal K was found to function against Enterococcus. faecalis, Lactobacillus plantarum and Streptococcus sp (Sangtanoo et al., 2014). Circular dichroism study of Sal K peptide revealed unordered structure in This-Cl buffer solution, ph 9.0, at 25. However, Sal K showed α-helical structure in 10 % sodium dodecyl sulfate (SDS), ph 9.0, at 25 C and presented β- sheet in 2 mm dodecylphosphocholine (DPC) micelles and liposome, ph 9.0, at 25 (Sangtanoo et al., 2014). Alb β showed complete amino acid homology to bacteriocin abp118-β from L. salivarius ABP118 (Pilasombut, 2006). Alb β peptide is also heat tolerant and inhibits some food-spoilage pathogens, such as L. sakei, Listeria innocua and Brochotrix campestris (Pilasombut, 2006). Alb β displayed α-helical structure in 10 % SDS and presented presented β-sheet in 2 mm DPC micelles and liposome, ph 9.0, at 25 (Sangtanoo et al., 2014). However, there is no further structural study of Sal K and Alb β have been reported and the interaction of Sal K and Alb β has not been reported. Nevertheless, the mode of action of these peptides is still unclear. Hence, to gain more in their structural information and their mode of action, we aimed to characterize secondary structure of these peptides such as circular dichroism (CD), nuclear magnetic resonance (NMR) and isothermal calorimeter (ITC). It had been reported that AMPs interacted to membrane of bacteria, and adopted secondary structure (Bonucci et al., 2013 and Morisset et al., 2004). After binding, the AMPs damaged cell membrane, resulting in the leakage of essential molecules such as ions, amino acids and ATP lead to cell dead. To investigate the interaction between peptides and membrane, the membrane-mimiking reagents, such as DPC, SDS or liposome has been used to create membrane mimicking environment. The zwitterionic micelles created by DPC representing an eukaryotic cell membrane while the prokaryotic cell membrane was created by anionic micelles (10% SDS). In this study, we investigate peptides conformation in two micellar systems However, in this report we showed only CD result of Sal K, Alb β and mixture of two peptides in different conditions, including unbuffered water, 2 mm DPC, ph 4.0, and 10% SDS, ph 4.0. After that, peptides in each buffer conditions will be performed by NMR to characterize their structures in detail, and the mode of action of these peptides will be further investigated. Due to Sal K and Alb β are produced from Lactobacillus sp. and its antimicrobial property against many food-spoilage bacteria. Therefore,

3 understanding the secondary structure and mode of action of Sal K and Alb β may be applied to food in order to protect food from food pathogens. 1. Peptide synthesis MATERAILS AND METHODS Sal K and Alb β peptides without any modification at their N- or C- terminus were synthesized by Biomatik, USA. Each peptide contains 47 amino acids. The peptides were purified by HPLC, and their sequences were confirmed by MS analysis. The amino acid sequences of Sal K and Alb β were; KRYPNCTGKFLGGLAKGAALGAISGGGVPGAVIGGNIGMVAGAISCL and KNGYGGSGIRWVHCGAGIVSGALMGSIGGNAWGAVAGGISGGIKSCR, respectively. 2. Characterization of antimicrobial peptide by Circular dichroism (CD) In order to characterize the secondary structures of sal K and Alb β peptides in different buffer conditions, the CD spectra of peptides were measured by J-815 spectropolarimeter (Jasco, Japan). The buffers used in this study were unbuffered water, 2 mm DPC, ph 4.0, and 10% SDS, ph 4.0. The experiments were performed in 3 individual sets; (i). Sal K, Alb β and mixture of Sal K and Alb β in unbuffered water, (ii). Sal K, Alb β and mixture of Sal K and Alb β in 2 mm DPC, ph 4.0, (iii). Sal K, Alb β and mixture of Sal K and Alb β in 10% SDS, ph 4.0. For each experiment, 0.1 mg of each peptide was dissolved in water, ph 4.0, 2 mm DPC, ph4.0 and 10% SDS, ph 4.0. However, for peptide mixture experiment, the 0.05 mg of each peptide was used in order to decrease the electrical conductivity of the CD spectrometer equipment. The CD spectra were measured in 1-nm path length quartz cell at 25 C. The spectra were recorded from nm, and the scanning speed was 50 nm/min. Each spectrum was the average from 3 scans. The spectra that appeared at very high voltage were eliminated to get very precise result. The secondary structure content of each peptide from CD spectra was predicted by K2D3 web server (

4 RESULTS AND DISCUSSION Secondary structure of antimicrobial peptides Sal K and Alb β In order to characterize secondary structure of antimicrobial peptides,sal K and Alb β, the CD spectra of the peptides were measured in different conditions;, which were unbuffered water, anionic micelles (10% SDS) and zwitterionic micelles (2 mm DPC). After CD spectra measurement, the secondary structures of the peptides were predicted by K2D3 web server (Bonucci et al., 2013). In this study, we found that Sal K and Alb β peptides were unstructured which were corresponded to other publications, but it showed 30.32% and 40.48% of α-helicity, respectively (Figure 1A and Table 1) which corresponded to many studies of other AMPs in solution that showed most of short peptides were unstructured in aqueous solution such as Lactococcin G (Hauge et al, 1998). Maculatin 1.1 (Sani et al., 2013) and Mesentericin Y105 (Morisset et al., 2004), Moreover, we found an unexpected result that the peptide mixture adopted helical conformation with α-helicity of 52.70%. However, there has no study of the secondary structure formation of peptide mixtures has reported before. This indicates that the mixing of Sal K and Alb β might interact and induces conformational change in unbuffered water. Moreover, the helical structure in aqueous condition was unusual. Therefore, to investigate the interaction between Sal K and Alb β, other methods such as ITC or NMR will be performed. In zwitterionic micelles or in 2 mm DPC, the Sal K peptide showed helical content of % while the Alb β peptide was unstructured. However, the Sal K showed β-sheet in 2 mm DPC at ph 9.0. This might conclude that secondary structure of Alb β is ph dependent. CD spectra characterization study of lactococcin G, which is the two-peptide bacteriocins containing LcnG-α and LcnG-β from Lactococcus lactis showed increase in α-helicity in DPC micelles (Hauge et al., 1998). The percentage of α-helix and β-sheet structure of the mixture peptide were; 38.71% and 17.54%, respectively (Figure 1B and Table 1). The result revealed that the Sal K peptide might interact with the micelles and formed helical conformation, but the helical content was decreased in peptide mixture. It might be conclude that the combination of two peptides in 2 mm DPC might reduce the helicilty due to the adding of unstructured Alb β peptide. In anionic micelles or 10% SDS, the Sal K, Alb β and the peptide mixture dramatically increased the helical content, which were 94.61%, 94.11% and 91.03%, respectively. (Figure 1C and Table 1). This result indicated that the peptides interacted with the SDS micelles, and adopt helical conformation in 10% SDS which corresponds to the result that performed by Sangtanoo et al., 2014 at different ph 9.0.

5 A AU sal K alb β -20 Mixture Wavelength (nm) B Sal K Alb β Mixture AU Wavelength (nm) C Sal K Alb β Mixture AU Figure 1. CD spectra of anitimicrobial peptide Sal K, Alb β and peptide mixture. The peptides were prepared in various buffer conditions, water ph 4.0 (A), 2 mm DPC ph 4.0 (B) and 10% SDS ph 4.0 (C) *AU is arbitrary unit Wavelength (nm)

6 Table 1. Percentage of secondary structure of Sal K and Alb β peptides in various buffer conditions as predicted by K2D3 web server Peptide Secondary structure content Water 2 mm 10% SDS DPC Sal K α-helix β-sheet Unstructured Alb β α-helix β-sheet Unstructured Peptide mixture α-helix β-sheet Unstructured CONCLUSION Structural characterization of Sal K and Alb β from L. salivarius K4 in various buffer conditions revealed different conformations of peptides. In the unbuffered water, the Sal K and Alb β were unstructured, but combination of Sal K and Alb β and peptide mixture revealed helical structure. In 2 mm DPC, only the Sal K peptide adopted α-helical structure while Alb β and the peptide mixture were unstructured. However, in 10% SDS, each peptide and the peptide mixture adopted high percentage of α-helicity. ACKNOWLEDGEMENTS This work was supported by Center for Advanced Studies in Tropical Natural Resources, National Research University-Kasetsart University, Kasetsart University, Chatuchak, Bangkok, 10900, Thailand (CASTNAR, NRU-KU, Thailand)

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