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Monitoring Antimicrobial Mechanisms of Surface Immobilized Peptides in Situ Minyu Xiao, Joshua Jasensky, Leanna L. Foster, Kenichi Kuroda, and Zhan Chen Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.7b03668 • Publication Date (Web): 15 Jan 2018 Downloaded from http://pubs.acs.org on January 15, 2018

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Monitoring Antimicrobial Mechanisms of Surface Immobilized Peptides in Situ Minyu Xiao1†, Joshua Jasensky1†, Leanna Foster2, Kenichi Kuroda1,2,3, Zhan Chen*,1,2 1

Department of Chemistry, University of Michigan, Ann Arbor, Michigan, 48109, USA

2

Macromolecular Science and Engineering Center, University of Michigan, Ann Arbor,

Michigan 48109, USA 3

Department of Biologic and Materials Sciences, School of Dentistry, University of Michigan,

Ann Arbor, Michigan 48109, USA

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Abstract: Antimicrobial peptides (AMPs) free in solution can kill bacteria by disrupting bacterial cell membranes. Their modes of action have been extensively studied and various models ranging from Pore formation to Carpet-like mechanism were proposed. Surface immobilized AMPs have been used as coatings to kill bacteria and as sensors to capture bacteria, but the interaction mechanisms of surface immobilized AMPs and bacteria are not fully understood. In this research, an analytical platform, SFG-microscope, which is composed of a sum frequency generation (SFG) vibrational spectrometer and a fluorescence microscope, was used to probe molecular interactions between surface immobilized AMPs and bacteria in situ in real time at the solid/liquid interface. SFG probed the molecular structure of surface immobilized AMPs while interacting with bacteria, and fluorescence images of dead bacteria were monitored as a function of time during the peptide-bacteria interaction. It was believed that upon bacteria contact, the surface immobilized peptides changed their orientation and killed bacteria. This research demonstrated that the SFG-microscope platform can examine the structure and function (bacterial killing) at the same time in the same sample environment, providing in-depth understanding on the structure-activity relationships of surface immobilized AMPs.

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Introduction

Medical device caused healthcare associated infections (HAIs) increased rapidly in the United States, with 1.7 million cases occurred in a recent year.1 Such infections could happen because of bacterial adherence, growth and proliferation on the surfaces of medical devices, and the contact with and implantation of such medical devices into patients’ body. One approach to decrease the HAIs incidence is to use antimicrobial surfaces for medical devices to prevent bacteria from adhering or kill bacteria that were already adhered.2 Conventional antimicrobial approaches include the use of antibiotics; however, many bacteria could develop drug resistance against such traditional antibiotics. Recently extensive research has been performed to use surface immobilized antimicrobial peptides (AMPs) as antimicrobial coatings. AMPs exhibit excellent activity in the killing of bacteria and superior biocompatibility (not kill mammalian cells). Molecular antimicrobial mechanisms of AMPs against bacteria in free solution have been extensively studied and several modes of action of AMPs have been proposed.3-6 It is well believed that AMPs’ functions are influenced by the peptide charge, conformation, hydrophobicity and amphipathicity. However, the antimicrobial mechanisms for surface immobilized AMPs have not been studied, due to the lack of appropriate tools to probe AMP-bacteria interactions. Sum frequency generation (SFG) vibrational spectroscopy is a second order nonlinear optical spectroscopy with submonolayer surface specificity.7-28 SFG has been applied to investigate many surfaces and interfaces to elucidate molecular structures of many molecules such as biological molecules like peptides and proteins, polymers, and water at interfaces.7-28 SFG has also been used to probe molecular structures of surface immobilized AMPs. Effects of peptide surface immobilization sites (e.g., via C- or N-terminus), chemical environments (e.g., 3 ACS Paragon Plus Environment

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air vs. water), immobilization strategies, and surface hydrophobicity on surface immobilized AMP conformation and orientation have been elucidated by SFG as well as supplemented molecular dynamics simulation studies.2, 29-32 However, the in-situ interactions between surface immobilized AMPs and live bacteria have not been investigated using SFG. Here, using an analytical platform with an SFG spectrometer and a fluorescence microscope (microscopeSFG),33-34 for the first time we monitored the AMPs’ behavior on surface while interacting with bacterial cell surfaces, and followed the antimicrobial action of peptides at the same time.

Materials and Methods Antimicrobial peptide surface preparation: Here we studied molecular interactions between surface immobilized MSI-78 and live E. coli BL21 (DE3) cells. MSI-78 is a synthetic analogue of the most widely studied AMP family magainin 2. MSI-78 has been reported to adopt alpha helical structure upon binding to bacterial cell membranes, as well as when immobilized onto a substrate surface without any interaction with bacterial cell membranes,2 which makes it an ideal candidate for SFG structural characterization. MSI-78 (sequence: GIGKFLKKAKKFGKAFVKILKK) used in this work was purchased from Peptide 2.0 and used as received. The MSI-78 peptide molecules were covalently immobilized onto a self-assembled monolayer terminated with maleimide groups on a CaF2 prism surface with silica coating. The immobilization was carried out via the thiol-maleimide linkage between the thiol group of the Nterminus cysteine modified MSI-78 peptide and the maleimide terminated SAM. The SAM was prepared on a silica surface via a two-step procedure: A silica coated (200nm) right-angle CaF2 prism was placed in an alkyne terminated silane solution for 24 h to prepare an alkyne terminated

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SAM. This SAM was then immersed in a 120 mM azido-PEG3-maleimide linker (Click Chemistry Tools, AZ107-10) solution (prepared by mixing 25 mg linker kits in 560mL DMSO for 1h, followed by dissolving into 20 mL pH 8.0 phosphate buffer at a concentration of 50 mM). The above reaction was catalyzed with 100 μM copper (II) and reducing agent (+)-sodium Lascorbate for 12 hours at room temperature. The prepared maleimide surface was then immersed in a MSI-78 peptide solution (1:1 TCEP premixed to break inter-peptide disulfate bond) with a concentration of 5 μM. Bacteria activity testing: We used the LIVE/DEAD™ BacLight™ Bacterial Viability Kits to perform the bacteria testing using E. coli BL21 (DE3). The dye concentration of 0.6μL of the component A and B each (five times dilution compared to the suggested amount) was used in the experiment to minimize the effect of the dyes on the bacteria activity. To allow a maximum amount of bacteria to be stained, the bacteria were stained for 15 min prior to test. The microscope-SFG experiments were performed by injecting 20 μL of the stained bacteria solution onto the rightangle CaF2 prism with immobilized peptides. The sample was covered with a cover slip to minimize the water evaporation during the experiment. Microscope SFG: SFG theory and instrumentation have been extensively reported in the literature7-9 and will not be repeated here. Details of the microscope-SFG has been published.34 Briefly, we designed our apparatus to allow simultaneous observation of peptide-bacteria cell interactions by florescent microscopy and SFG spectroscopy. To allow the fluorescence microscope to fit into the SFG sample stage, an “inverted” SFG sample geometry was used. Both the 532 nm visible photon and the frequency tunable infrared photon were spatially and temporally overlapped onto

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the CaF2 prism surface. The facing-up CaF2 prism was placed on a translational stage, which could be adjusted or moved in all three dimensions, allowed the sample for imaging with the fluorescent microscope. A telescope, filter set and a CCD camera were placed on top of the prism surface for imaging the sample. A picture of the microscope-SFG setup can be found in Figure S1. While SFG can monitor the structure of the surface immobilized AMPs when interacting with the bacteria, the fluorescent microscope can provide snapshots of bacterial live/dead information at given time intervals. SFG spectra and fluorescent images were taken from the sample during the peptidebacteria interactions. When the fluorescent images were taken, SFG PMT detector was shut to minimize the interference of the fluorescent excitation light to the SFG signal. Fluorescent images were taken at multiple time points so that there are blank spots on the SFG timedependent signal curve.

Results and Discussion:

Figure 1 (a) SFG ppp and ssp spectra and the fitted results of surface immobilized MSI-78 in contact with water; (b) time-dependent observation of ppp signal at 1650 cm-1 after the surface immobilized MSI-78 in contact with E-coli solution; (c) SFG ppp and ssp spectra and fitted

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results of surface immobilized MSI-78 while interacting with bacteria (collected after surface immobilized MSI-78 in contact with E-coli solution for 1 hour). Surface immobilized AMP structure (e.g., conformation or orientation) can be probed by SFG amide I signal. It has been demonstrated that the SFG peak centered near 1650 cm-1 originates from alpha helical structure in peptide/protein.35 By fitting the ssp (s-polarized SFG signal, s-polarized input visible beam, and p-polarized input IR beam) and ppp SFG amide I signals collected from the peptides, χppp and χssp could be measured. By taking the χppp/χssp ratio, tilt angle θ of an interfacial alpha helical structure can be then deduced. (θ is described as the angle between the surface normal and the main axis of the target alpha helical structure). Detailed correlation of alpha helical structure orientation and SFG signal strength ratio has been published previously.35-37 Figure 1a shows the SFG ppp and ssp amide I spectra collected from surface immobilized MSI-78 in an aqueous environment before contacting with bacteria. The deduced χppp/χssp ratio from the fitting results of the SFG spectra is 1.62. According to the relationship between the measured χppp/χssp ratio of an alpha-helical peptide and the peptide orientation (Figure 2), the orientation of surface immobilized MSI-78 could be determined to be around 10o, indicating that the alpha helical structure of the N-terminus immobilized MSI-78 adopts a standing-up orientation on the surfaces. Figure 1b shows the time-dependent SFG ppp signal intensity observed at 1650 cm-1 after adding E. coli onto the immobilized AMP surface. The SFG timedependent result shows that the SFG ppp signal intensity at 1650 cm-1 decreased as a function of time upon adding E. coli to the solution in contact with surface immobilized peptides, and reached a plateau after 3000 s. Both the orientation and number of surface immobilized MSI-78 molecules could affect the observed SFG signal intensity. Here the peptides were chemically

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immobilized on the SAM surface on silica; the peptide coverage on SAM was unlikely changed after bacteria contact. We therefore decided to measure the surface immobilized peptide orientation after the signal stabilized to see how the peptide-bacteria interaction could change the peptide orientation. Figure 1c shows the ppp and ssp spectra collected from the surface immobilized MSI-78/bacteria solution interface 3000 s after the initial peptide-bacteria solution contact. The fitted SFG results indicated that the measured χppp/χssp ratios for surface immobilized peptides in buffer (before bacteria contacting) and in contact with bacteria for 3000 s are markedly different (before: 1.62, after: 1.16). This clearly indicated that the surface immobilized AMP had a structural (e.g., orientation) change upon contacting E-coli bacteria. Based on the correlation between the χppp/χssp ratio and the alpha helix tilt angle θ (Fig 2), it is impossible to find a tilt angle θ which could satisfy the measured χppp/χssp ratio of the surface immobilized MSI-78 after contacting E-coli; the ratio is lower than the lowest possible ratio in the curve. It is worth mentioning that the correlation plotted in Fig.2 was deduced under the delta orientation angle assumption. That is, all the MSI-78 molecules are assumed to adopt the same tilt angle. As we published previously, for a Gaussian distribution, the lowest possible χppp/χssp ratio is higher than that of a delta distribution. Therefore here the measured χppp/χssp ratio of surface immobilized MSI-78 after contacting E-coli could not be described by a Gaussian orientation function as well.35 As we published previously, such a measured ratio likely indicates a multiple orientation distribution of alpha helical peptides. For example, such a ratio may be caused by a peptide orientation distribution with two orientation angles with different absolute (up or down) orientations of alpha helical peptides.35 Our observation here could be interpreted by the existence of two types of surface immobilized MSI-78 peptides after contacting the bacteria

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solution: Some of the peptides were not interacting with bacteria, therefore they adopt the original standing up pose. The rest of the peptides interacting with the bacteria could bend towards the surface. For both cases, we assume that they still adopt alpha-helical structures. Unfortunately, it is impossible to characterize the detailed structure of the “bent” peptides after interacting with the bacteria, instead, here we assume that the peptides contacting with bacteria point to a different absolute direction. With such a picture and based on the two independent measurements (the measured ppp/ssp SFG signal strength ratio from peptides in contact with bacteria and the signal strength ratio of the SFG spectra from the peptides before and after bacteria contact) as well as the orientation of the peptides before bacteria contact, we could deduce two unknowns: (1) the ratio of the peptides not contacting vs. contacting bacteria of ~1.5 (60% not contacting, 40% contacting); (2) the orientation of the peptides in contact with bacteria: the χppp/χssp ratio of the second MSI-78 pose was calculated to be 2.04, showing the orientation angle is about ~110o vs. the surface normal. Figure 2 shows the signal contributions of the two types of peptides to the overall observed spectra collected from the surface immobilized peptide/bacteria solution interface as well as the schematic showing the peptide orientations. Details of the spectra fitting parameters and formulas used for above quantitative deductions are presented in the supporting information. Basically, there are two equations and two unknowns, so we can solve both unknowns. From the optical image of the bacteria deposited surface (supporting information), we can see that bacteria cover slightly less than 50% of the surface, showing that the above SFG conclusion is reasonable. SFG results provided insightful orientation information of the surface immobilized MSI78 while interacting with E-coli. As discussed above, MSI-78 is antimicrobial peptide. Its sequence is GIGKFLKKAKKFGKAFVKILKK, with 9 cationic lysine residues. The modes of

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antimicrobial actions of many AMPs free in solution have been extensively examined.3-6 Different modes such as Barrel-stave Pore model and Toroidal Pore model in which peptides form pores in the bacteria cell membrane or Carpet Model and Detergent Model in which the AMPs adhere and lie down onto the cell membrane.3-6 Among all different models proposed, the electrostatic force plays a vital role.38-40 The positive charged AMPs have strong attractive forces with negatively charged bacteria cell membrane, therefore AMPs can strongly adhere to the bacteria cell member for action.

Figure 2 (a, b) Contributions of the PPP and SSP signals of two types of MSI-78 peptides at the surface immobilized peptides/E-coli solution interface. The black lines are the fitting results of the collected SFG spectra shown in Figure 1 from the surface immobilized peptides/E-coli solution interface. The red (up - from peptides not contacting with bacteria) and blue (down from peptides contacting with bacteria) spectra are the spectra contributed from the peptides with different orientations. Each spectrum was plotted using χ(2). The positive and negative peaks indicate peptides with different absolute orientations. (c-1,2) Schematic of orientations of the two types of surface immobilized AMPs (not contacting bacteria and contacting bacteria). Black dashed line: surface normal; red arrow: alpha helical peptide principal axis.

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The mode of action of MSI-78 in free solution has been examined and published.41 We believe that the mode of action of MSI-78 in free solution could not be used to interpret surface immobilized peptide-bacteria interactions. The surface immobilized MSI-78 peptide has a length of 3.6 nm. Even if it stands on the surface vertically, it could not penetrate through the cell wall or outer cell membrane of bacteria. Therefore, they could not reach the bacteria inner cell membrane to form pores. For AMPs in free solution with either a Barrel-stave Pore model and a Toroidal Pore model, the ability of drilling a hole on membranes originates from AMPs’ freedom in solution. Here the surface immobilized AMPs do not have such freedom, and the length of MSI-78 does not allow it to fully extend into the bacteria inner cell membrane and create a hole. Therefore we believe that the mode of action of surface immobilized MSI-78 to kill bacteria is neither through Barrel-stave Pore model nor through Toroidal Pore model. Instead, here the charge interaction should play a major role in bacteria killing.

Figure 3 (a) Live E-coli; (b) Dead E-coli at 0min; (c) Dead E-coli at 20min; (d) Dead E-coli at 40min; (e) Dead E-coli 60min. Scale bar: 10μm.

Beside the above structure information of surface immobilized peptides while interacting with bacteria which we could extract from the SFG results, the antimicrobial behavior of surface immobilized AMP was also examined simultaneously by monitoring the E. coli killing process under a fluorescence microscope. 11 ACS Paragon Plus Environment

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To determine bacterial cell death, the E. coli cells adhered on the surface were monitored in the presence of propidium iodide (PI), which can enter into cells with damaged cell membranes and stain the cells by binding to DNA molecules inside the cells. Therefore, the red fluorescence signal detected from the bacterial cells reflects the disruption of the inner (cytoplasmic) membrane of these E. coli cells. To minimize the impact of dye molecules to the E. coli cells and potentially the SFG signal contribution from dye molecules, we only partially stained the E. coli with small amount of dye (five times dilution compared to the suggested concentration – there should be more bacteria on the surface. Figure S3 in the supporting information shows the E-coli amount on the prism surface). Figure 3a-e showed live E. coli and dead E. coli on surface immobilized MSI-78 at different time points. Fluorescence images showed that only very few bacteria were dead in the first 20 min. More bacteria began to die starting from 40 min, and significant number of bacteria died after 60 min. The fluorescence activity images measured show that the bacteria could be killed by the surface immobilized peptide contacting. Such contacting varied the peptide orientation, leading to SFG signal intensity decrease. The time-dependent SFG signal change can be interpreted in two ways: (1). The bacteria attached to the surface quickly, but the bacteria killing was slow. The peptides slowly bended to kill bacteria or after the bacteria were killed, the peptides changed orientation, leading to SFG signal decrease. (2) The bacteria attached to the surface slowly, but the bacteria killing process was fast. The orientation change of peptides quickly happened when in contact with the bacteria to kill bacteria. Then the slow SFG signal time dependent change was caused by the slow adhesion rate of bacteria to the surface.

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The current fluorescence assay does not provide enough evidence on differentiating the above two possibilities, but some previous studies suggested that even though factors like surface roughness, surface charge, and bacterial strain might affect adhesion of bacteria onto a surface, the overall rate of bacteria deposition onto a surface was not fast, could be hours.42-44 Therefore we believe the above possibility (2) is better for us to use to explain the SFG time-dependent results: The bacteria slowly attached to a surface, interacting with the peptides. The peptides changed orientations and killed the bacteria due to the charged interactions.

Conclusion: To conclude, combining SFG with fluorescent microscopy, the in-situ observation on structure and activity of surface immobilized AMPs interacting with bacteria has been achieved. Upon bacteria contact, the surface immobilized peptides changed orientation (likely to a bent structure pointing to the other direction) and kill bacteria. We believe that this is the first time to measure the structure and function of surface immobilized peptides while interacting with live bacteria in situ at the same time to understand the structure-function relationships of surface immobilized AMPs, which is important for future development of surface immobilized AMPs with improved performance.

Supporting Information: Procedure of surface immobilized MSI-78, detailed peak fitting parameters. This material is available free of charge via the Internet at http://pubs.acs.org.

Author Information

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Corresponding Author *E-mail: [email protected]

Notes M.X. and J.J contributed equally to this work. The authors declare no competing financial interest.

Acknowledgement This research is supported by the National Science Foundation (CHE-1505385 for ZC) and the University of Michigan.

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18. Yan, E. C.; Fu, L.; Wang, Z.; Liu, W., Biological macromolecules at interfaces probed by chiral vibrational sum frequency generation spectroscopy. Chem. Rev. 2014, 114 (17), 8471-8498. 19. Adhikari, N. M.; Premadasa, U. I.; Cimatu, K. L., Sum frequency generation vibrational spectroscopy of methacrylate-based functional monomers at the hydrophilic solid–liquid interface. Phys. Chem. Chem. Phys. 2017, 19 (32), 21818-21828. 20. Premadasa, U. I.; Adhikari, N. M.; Baral, S.; Aboelenen, A. M.; Cimatu, K. L. A., Conformational Changes of Methacrylate-Based Monomers at the Air–Liquid Interface Due to Bulky Substituents. J. Phys. Chem. C 2017, 121 (31), 16888-16902. 21. Tan, J.; Zhang, B.; Luo, Y.; Ye, S., Ultrafast Vibrational Dynamics of Membrane‐ Bound Peptides at the Lipid Bilayer/Water Interface. Angew. Chem. 2017, 129 (42), 1315713161. 22. Van der Post, S. T.; Hsieh, C. S.; Okuno, M.; Nagata, Y.; Bakker, H. J.; Bonn, M.; Hunger, J., Strong frequency dependence of vibrational relaxation in bulk and surface water reveals sub-picosecond structural heterogeneity. Nat. Commun. 2015, 6, 8384. 23. Lis, D.; Backus, E. H.; Hunger, J.; Parekh, S. H.; Bonn, M., Liquid flow along a solid surface reversibly alters interfacial chemistry. Science 2014, 344, 1138-1142. 24. Okur, H. I.; Kherb, J.; Cremer, P. S., Cations bind only weakly to amides in aqueous solutions. J. Am. Chem. Soc. 2013, 135 (13), 5062-5067. 25. Troiano, J. M.; McGeachy, A. C.; Olenick, L. L.; Fang, D.; Liang, D.; Hong, J.; Kuech, T. R.; Caudill, E. R.; Pedersen, J. A.; Cui, Q.; Geiger, F. M., Quantifying the Electrostatics of Polycation–Lipid Bilayer Interactions. J. Am. Chem. Soc. 2017, 139 (16), 5808-5816. 26. Xiao, M.; Joglekar, S.; Zhang, X.; Jasensky, J.; Ma, J.; Cui, Q.; Guo, L. J.; Chen, Z., Effect of interfacial molecular orientation on power conversion efficiency of perovskite solar cells. J. Am. Chem. Soc 2017, 139 (9), 3378-3386. 27. Hsieh, C.-S.; Okuno, M.; Hunger, J.; Backus, E. H. G.; Nagata, Y.; Bonn, M., Aqueous Heterogeneity at the Air/Water Interface Revealed by 2D-HD-SFG Spectroscopy. Angew. Chem. International Edition 2014, 53 (31), 8146-8149. 28. Wang, J.; Even, M. A.; Chen, X.; Schmaier, A. H.; Waite, J. H.; Chen, Z., Detection of amide I signals of interfacial proteins in situ using SFG. J. Am. Chem. Soc. 2003, 125 (33), 9914-9915. 29. Wei, S.; Zou, X.; Cheng, K.; Jasensky, J.; Wang, Q.; Li, Y.; Hussal, C.; Lahann, J.; Brooks III, C. L.; Chen, Z., Orientation Determination of a Hybrid Peptide Immobilized on CVDBased Reactive Polymer Surfaces. J. Phys. Chem. C 2016, 120 (34), 19078-19086. 30. Wang, Q.; Wei, S.; Wu, J.; Zou, X.; Sieggreen, O.; Liu, Y.; Xi, C.; Brooks III, C. L.; Chen, Z., Interfacial Behaviors of Antimicrobial Peptide Cecropin P1 Immobilized on Different SelfAssembled Monolayers. J. Phys. Chem. C 2015, 119 (39), 22542-22551. 31. Wang, Z.; Han, X.; He, N.; Chen, Z.; Brooks III, C. L., Molecular structures of C-and Nterminus cysteine modified cecropin P1 chemically immobilized onto maleimideterminated self-assembled monolayers investigated by molecular dynamics simulation. The J. Phys. Chem. B 2014, 118 (21), 5670-5680. 32. Han, X.; Liu, Y.; Wu, F.-G.; Jansensky, J.; Kim, T.; Wang, Z.; Brooks, C. L.; Wu, J.; Xi, C.; Mello, C. M.; Chen, Z., Different Interfacial Behaviors of Peptides Chemically Immobilized on Surfaces with Different Linker Lengths and via Different Termini. The J. Phys. Chem. B 2014, 118 (11), 2904-2912. 16 ACS Paragon Plus Environment

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33. Zhang, C.; Jasensky, J.; Wu, J.; Chen, Z. In Combining surface sensitive vibrational spectroscopy and fluorescence microscopy to study biological interfaces, SPIE BiOS, International Society for Optics and Photonics. 2014, pp 894712-894712-8. 34. Zhang, C.; Jasensky, J.; Leng, C.; Del Grosso, C.; Smith, G. D.; Wilker, J. J.; Chen, Z., Sum frequency generation vibrational spectroscopic studies on buried heterogeneous biointerfaces. Opt. Lett. 2014, 39 (9), 2715-2718. 35. Nguyen, K. T.; Le Clair, S. V.; Ye, S.; Chen, Z., Orientation determination of protein helical secondary structures using linear and nonlinear vibrational spectroscopy. The J. Phys. Chem. B 2009, 113 (36), 12169-12180. 36. Yang, P.; Boughton, A.; Homan, K. T.; Tesmer, J. J.; Chen, Z., Membrane orientation of Gαiβ1γ2 and Gβ1γ2 determined via combined vibrational spectroscopic studies. J. Am. Chem. Soc. 2013, 135 (13), 5044-5051. 37. Liu, Y.; Ogorzalek, T. L.; Yang, P.; Schroeder, M. M.; Marsh, E. N. G.; Chen, Z., Molecular orientation of enzymes attached to surfaces through defined chemical linkages at the solid– liquid interface. J. Am. Chem. Soc. 2013, 135 (34), 12660-12669. 38. Radzishevsky, I. S.; Rotem, S.; Bourdetsky, D.; Navon-Venezia, S.; Carmeli, Y.; Mor, A., Improved antimicrobial peptides based on acyl-lysine oligomers. Nat. Biotechnol. 2007, 25 (6), 657-659. 39. Vaidyanathan, S.; Chen, J.; Orr, B. G.; Banaszak Holl, M. M., Cationic polymer intercalation into the lipid membrane enables intact polyplex DNA escape from endosomes for gene delivery. Mol. Pharm. 2016, 13 (6), 1967-1978. 40. Vaidyanathan, S.; Orr, B. G.; Banaszak Holl, M. M., Role of cell membrane–vector interactions in successful gene delivery. Acc. Chem. Res. 2016, 49 (8), 1486-1493. 41. Hallock, K. J.; Lee, D.-K.; Ramamoorthy, A., MSI-78, an analogue of the magainin antimicrobial peptides, disrupts lipid bilayer structure via positive curvature strain. Biophys. J. 2003, 84 (5), 3052-3060. 42. Mafu, A. A.; Plumety, C.; Deschênes, L.; Goulet, J., Adhesion of pathogenic bacteria to food contact surfaces: Influence of pH of culture. Int. J. Microbiol. 2010, 2011. 10 43. Otto, K.; Silhavy, T. J., Surface sensing and adhesion of Escherichia coli controlled by the Cpx-signaling pathway. Proc. Natl. Acad. Sci. 2002, 99 (4), 2287-2292. 44. Terada, A.; Yuasa, A.; Kushimoto, T.; Tsuneda, S.; Katakai, A.; Tamada, M., Bacterial adhesion to and viability on positively charged polymer surfaces. Microbiology 2006, 152 (12), 3575-3583.

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Figure 1 (a) SFG ppp and ssp spectra and the fitted results of surface immobilized MSI-78 in contact with water; (b) time-dependent observation of ppp signal at 1650 cm-1 after the surface immobilized MSI-78 in contact with E-coli solution; (c) SFG ppp and ssp spectra and fitted results of surface immobilized MSI-78 while interacting with bacteria (collected after surface immobilized MSI-78 in contact with E-coli solution for 1 hour). 323x86mm (150 x 150 DPI)

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Figure 2 (a, b) Contributions of the PPP and SSP signals of two types of MSI-78 peptides at the surface immobilized peptides/E-coli solution interface. The black lines are the fitting results of the collected SFG spectra shown in Figure 1 from the surface immobilized peptides/E-coli solution interface. The red (up - from peptides not contacting with bacteria) and blue (down - from peptides contacting with bacteria) spectra are the spectra contributed from the peptides with different orientations. Each spectrum was plotted using χ(2). The positive and negative peaks indicate peptides with different absolute orientations. (c-1,2) Schematic of orientations of the two types of surface immobilized AMPs (not contacting bacteria and contacting bacteria). Black dashed line: surface normal; red arrow: alpha helical peptide principal axis. 372x91mm (150 x 150 DPI)

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Figure 3 (a) Live E-coli; (b) Dead E-coli at 0min; (c) Dead E-coli at 20min; (d) Dead E-coli at 40min; (e) Dead E-coli 60min. Scale bar: 10µm. 317x54mm (150 x 150 DPI)

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