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Real time observation of antimicrobial polycation effects on Escherichia coli: adapting the carpet model for membrane disruption to quaternary copolyoxetanes Congzhou Wang, Olga Yu Zolotarskaya, Sithara S Nair, Christopher J. Ehrhardt, Dennis E. Ohman, Kenneth J. Wynne, and Vamsi K Yadavalli Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.5b04247 • Publication Date (Web): 06 Mar 2016 Downloaded from http://pubs.acs.org on March 7, 2016

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Real time observation of antimicrobial polycation effects on Escherichia coli: adapting the carpet model for membrane disruption to quaternary copolyoxetanes

Congzhou Wang,1 Olga Y. Zolotarskaya,1 Sithara S. Nair,1 Christopher J. Ehrhardt,2 Dennis E. Ohman,3,4 Kenneth J. Wynne, 1,§ Vamsi K. Yadavalli1,§

1

Department of Chemical and Life Science Engineering, 2 Department of Forensic Science,

Virginia Commonwealth University, Richmond, Virginia, USA, 3 Department of Microbiology and Immunology, VCU School of Medicine, and 4 McGuire Veterans Affairs Medical Center, Richmond, VA 23249

§ Address correspondence to: Vamsi K. Yadavalli, [email protected], Kenneth J Wynne: [email protected]

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ABSTRACT

Real-time atomic force microscopy was used for analyzing effects of the antimicrobial polycation copolyoxetane P[(C12)-(ME2Ox)-50/50], C12-50 on the membrane of a model bacterium, Escherichia coli (ATCC# 35218). AFM imaging showed cell membrane changes with increasing C12-50 concentration and time including nanopore formation and bulges associated with outer bacterial membrane disruption. A macroscale bactericidal concentration study for C12-50 showed a 4 log kill at 15 µg/mL with conditions paralleling imaging (1 h, 1x PBS, physiological pH, 25 °C). The dramatic changes from the control image to 1 h after introducing 15 µg/mL C12-50 are therefore reasonably attributed to cell death. At the highest concentration (60 µg/mL) further cell membrane disruption results in leakage of cytoplasm driven by detergent-like action. The sequence of processes for initial membrane disruption by the synthetic polycation C12-50 follows the carpet model posited for antimicrobial peptides (AMPs). However, the nanoscale details are distinctly different as C12-50 is a synthetic, water soluble copolycation that is best modeled as a random coil. In a complementary AFM study, chemical force microscopy shows that incubating cells with C12-50 decreased the hydrophobicity across the entire cell surface at an early stage. This finding provides additional evidence indicating that C12-50 polycations initially bind with the cell membrane in a carpet-like fashion. Taken together, real time AFM imaging elucidates the mechanism of antimicrobial action for copolyoxetane C12-50 at the single cell level. In future work this approach will provide important insights into structure-property relationships and improved antimicrobial effectiveness for synthetic amphiphilic polycations.

Keywords: Copolyoxetane, Antimicrobial, Escherichia coli, Atomic force microscopy, Carpet model


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INTRODUCTION

Bacterial resistance to conventional antibiotics continues to emerge as a serious, worldwide threat to public health.1 For example, strains against β-lactam antibiotics have continuously evolved with a nearly 1000-fold increased resistance over the past three decades.2 Simultaneous development of resistance to several classes of antibiotics has created multidrug-resistant bacterial strains or “superbugs”.3 Due to the development of relatively few new antibiotics in recent years,4 studying different classes of antimicrobials to combat bacterial infections is of fundamental and applied importance. Previous studies in our group have shown promise for a novel class of copolyoxetanes as highly effective antimicrobials against Gram positive and negative bacteria. These water soluble copolyoxetanes with quaternary (quat) and PEG-like “ME2” side chains (Scheme 1) have low cytotoxicity for human red blood cells and dermal and foreskin fibroblasts.5, 6 C12-50, 1, was chosen for the present study as it is one of the “high performance” copolyoxetanes having these exciting properties. The highly effective biocidal activity of copolyoxetane 1 and congeners is of special interest, as in contrast to conventional antibiotics, negligible buildup of resistance has been observed for polycations.7, 8 Although structurally quite different, the amphiphilic nature of copolyoxetane 1 has a physical relationship to antimicrobial peptides (AMPs), which have received much attention.9, 10, 11, 12, 13, 14, 15

Several concepts have been proposed for the interaction of antimicrobial peptides with the

bacterial membrane including barrel stave,16, 17 toroidal pore18, 19 and carpet models.20, 21 These models describe processes associated with an antimicrobial peptide attaching to and inserting into cell membrane bilayers to form pores leading to its disruption and subsequent cell death. The elucidation of AMP biocidal effectiveness led to work on polycations with the goal of mimicking antimicrobial effectiveness and cytocompatibility.7, 22, 23, 24 Paralleling naturally occurring AMP polycations, the mechanism of bacterial kill for synthetic amphiphilic polycations is thought to involve membrane disruption primarily by ionic interactions between the polycation and bacterial membrane.25, 26 The net positive polycation charge drives ionic 3 ACS Paragon Plus Environment

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binding to negatively charged bacterial membrane surfaces, while an amphipathic structure facilitates insertion into the cell membrane.27, 28 Mechanisms for polycations and their effect on natural membrane systems, especially live bacteria are still in need of clarification. Thus, exploring synthetic polycation membrane disruption in situ has merit for elucidating the relationship of chemisorption to pore formation and osmotically driven processes leading to cell membrane destabilization. The challenge for direct study of polycation effects on natural membranes led to exploring direct imaging of cell lysis by atomic force microscopy (AFM). AFM has rapidly emerged as an important tool in microbiology and can be a powerful technique for studying mechanisms of biocidal agents on cells.29, 30, 31, 32 A unique advantage is the ability not only to visualize live cells under physiological conditions with nanoscale resolution and three-dimensional imaging, but also to interrogate cell surface biochemical properties such as hydrophobicity or specific biomarkers via functional probes.33, 34 As examples, chemical force microscopy (CFM) with hydrophobic -CH3 tips was used to probe local hydrophobic forces on Aspergillus fumigates and Mycobacterium bovis.35 AFM studies on bacterial cells have been reported, elucidating the mechanism of action of conventional antibiotics36, 37 and antimicrobial peptides.38, 39, 40, 41, 42, 43 However, in these investigations, different cells are usually removed from the medium at selected time points and imaged in air to obtain high resolution images. This results in cell drying that can alter morphology or create artifacts due to salts and compounds in the culture medium.40 A clearer insight into the effects of antimicrobial compounds on cells can be obtained by observations conducted in situ (fluid), and in real time so as to reveal the mechanisms of antimicrobial activity by imaging the cell surface. To date, relatively few investigations have involved imaging of bacterial changes due to antimicrobial treatments at the single cell level in liquid environments. E. coli surfaces influenced by the antimicrobial peptide CM15 were observed using high-speed AFM in real time.44 The single dividing Mycobacterium JLS was studied before and after anti-mycobacterial drug (ethambutol) treatment.45 The effect of antimycobacterial drugs (including isoniazid, ethionamide, ethambutol, and streptomycin) on Mycobacterium bovis BCG was studied using AFM imaging and CFM hyphophobicity mapping.46, 47 In this report, a strategy for understanding the mechanism of action of the synthetic 4 ACS Paragon Plus Environment

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polycation antimicrobial C12-50 1 is demonstrated via real time AFM imaging in fluid. Observations of nanoscale morphology and biochemical behavior of the cell surface are presented using live E. coli as the model bacterium. Importantly, the same cell is tracked over time to elucidate the mechanism of cell lysis by C12-50, Scheme 1.5, 6 In addition to observing cell morphology, preliminary results employing a functionalized AFM tip are provided that quantify the change in surface hydrophobicity attributed to chemisorption of C12-50. Imaging cell surface features at different C12-50 concentrations provides insight into mechanisms of antimicrobial action and supports the carpet model proposed for antimicrobial peptides.48 Detailed imaging and bacterial membrane response to C12-50 may provide a new avenue for optimizing design of synthetic amphiphilic polycations.

EXPERIMENTAL SECTION Materials and instrumentation Poly-L-lysine hydrobromide (P1524), Tris(hydroxymethyl) aminomethane and 1-undecanethiol were purchased from Sigma-Aldrich (St. Louis, MO). Phosphate-buffered saline, PBS pH 7.4, (11.9 mM phosphates, 137 mM sodium chloride and 2.7 mM potassium chloride) and ethanol (200-proof) were purchased from Fisher Scientific. Mica was purchased from Ted Pella (Redding, CA). Ultrapure water (resistivity 18.2 MΩ•cm) was obtained from a MilliQ water purification system (Millipore Scientific, MA). CSG30 cantilevers (NT-MDT) were used for non-contact imaging in liquid. Gold coated TR400PB cantilevers (Olympus) were used for chemical force spectroscopy in liquid. Cantilevers were cleaned using an UV/Ozone Procleaner (BioForce Nanosciences Inc. Ames, IA) before use. Imaging and chemical force mapping experiments were performed on an MFP-3D atomic force microscope (Asylum Research, Santa Barbara, CA).

Polymer synthesis and Characterization Copolyoxetane C12-50, 1 was synthesized as described earlier (Scheme 1).5, 6 Complete synthesis details and 1H-NMR are provided in the Supporting Information (Figures S1 and S2). 5 ACS Paragon Plus Environment

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A brief description follows. Monomers (a) 3-((2-(2-methoxyethoxy) ethoxy) methyl)-3methyloxetane (ME2Ox) and (b) 3-((4-bromobutoxy) methyl)-3-methyloxetane (BBOx) were used in ring opening co-polymerization to prepare P[(BBOx)0.48 (ME2)0.52. N,Ndimethyldodecylamine (1.5 mL) was added to a solution of P[(BBOx)0.48 (ME2)0.52 (1 g) in 10 mL of acetonitrile followed by heating at 72oC under nitrogen for 8 h. After removing the solvent under reduced pressure, the product was dried under vacuum overnight. To remove unreacted amine, 15 mL hexane was added to the product followed by heating at 50oC for 30 min. Hexane was decanted. This process was repeated with fresh hexane for a total of three times. Drying under reduced pressure for 24 h gave 1.4 g P[(C12)0.48 (ME2)0.52 designated C1250, which approximates the C12 segment mole fraction. C12-50 Mw ~ 7.7 kDa was estimated based on GPC of the precursor P[(BBOx)0.48 (ME2Ox)0.52 (Figure S3, Supporting Information). This approximation was supported by 1H-NMR that showed complete C12 amine substitution.

Sample preparation and atomic force microscopy (AFM) imaging E. coli (ATCC# 35218) were maintained on Trypticase Soy Agar (TSA) comprised of 30 g Trypticase soy broth (Becton Dickinson, Franklin Lakes, NJ) and 15 g agar (American BioAnalytical, Natick, MA). A freshly streaked plate was incubated overnight at 30°C. A single colony was picked and inoculated into 125 mL of Trypticase Soy Broth (TSB) (30 g Trypticase soy broth (Becton Dickinson, Franklin Lakes, NJ). The culture was incubated for 20 h at 30°C and 225 rpm in an orbital shaker. The bacterial mass was harvested by centrifuging at 3220 x g for 15 min, decanting the medium, and re-suspending the pellet in phosphate-buffered saline (PBS). Freshly cultured E. coli prepared as described above was used directly for AFM imaging. Growth medium was not employed as preliminary studies showed that tip chemisorption resulted in deteriorated images. Bacterial cells were immobilized on mica using a poly-L-lysine fixation method described earlier44 to increase the adherence of the bacteria. Briefly, freshly cleaved mica was immersed for 10 min in a solution of 0.05 mg/ml poly-L-lysine hydrobromide and 10 mM Tris (pH 8.0). The surface was then covered and dried while held vertically overnight at room temperature. The coated mica was stored at room temperature and was used within one week. 6 ACS Paragon Plus Environment

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Concentrated bacteria suspended in PBS were deposited on the coated mica and incubated for 30 min, taking care not to let the surface dry. Excess cells were rinsed off with three washes of 1 mL Millipore water. Images were taken in PBS with and without the antimicrobial copolyoxetane C12-50 using non-contact mode imaging. A series of concentrations (~15, 30 and 60 µg/mL) were used to study the concentration dependence of the action of copolyoxetane. To observe the bacteria in situ and in real time, the same cells (same areas on mica) were imaged at selected time intervals. Cell surface roughness was analyzed using Igor Pro 6.32 A (Wavemetrics Inc, OR).

AFM probe functionalization and chemical force mapping Gold coated cantilevers were cleaned in UV/ozone for 15 min. Cantilevers were functionalized via immersion in1-undecanethiol solution in ethanol for 16 h to obtain CH3 terminated group on probe surface. The cantilevers were rinsed with ethanol to remove unbound 1-undecanethiol. Subsequently, CH3-functionalized cantilevers with spring constant ~0.09 N/m and resonance frequency 32 kHz were used to obtain force data on cells in buffer. The same bacterial cells were force-imaged at selected time points (0 , 1.5 and 3 h). At least 5 different E. coli cells (from 3 different culture batches) were studied separately. Force-distance curves over the cell surface were obtained by collecting a series of sequential force curves in an m×n grid. Each force curve was obtained at the same loading rate (81 nN/s, at a ramp velocity of 900 nm/s) by pressing the cantilever to a low trigger point (200 pN), allowing binding to occur (contact time 0.1 s), and then retracting. All force maps were obtained by collecting ~32×32 force curves over a defined area (~2 µm × 2 µm), estimating the adhesion force values, and displaying these values by scale of color. The height maps of the same area were generated simultaneously with force mapping. All images including height and force maps were obtained using custom routines for fast data processing in Igor Pro 6.32 A (Wavemetrics Inc, OR).

Biocidal activity for copolyoxetane concentrations (15 - 60 µg/mL). To establish biocidal effectiveness for C12-50 against E. coli (ATCC# 35218) under conditions 7 ACS Paragon Plus Environment

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similar to those employed for AFM imaging (time, temperature, buffered environment), a conventional study of bacteriocidal effectiveness was carried out. An agar plate was prepared from BBL™ Trypticase soy agar, Cockeysville, MD and streaked with E. coli (ATCC# 35218). A single colony from this TSA plate was inoculated into 10 ml of TSB, which had been prepared from BD Bacto™ Trypticase Soy Broth, Sparks, MD. Incubation was carried out with shaking at 225 rpm overnight at 30 °C. The bacterial mass was then collected by centrifuging at 6000 rpm for 15 min, decanting the medium, and re-suspending the pellet in PBS. The optical density (O.D., 600 nm) was checked followed by dilution to 0.01. Based on calibration curves for previously employed E. coli strains, this O.D. corresponds to ~107 colony forming units per milliliter (CFU/mL). Four test tubes were prepared with 1 mL of the bacterial suspension. Appropriate aliquots of C12-50 stock solution (1 mg/mL) were added to give 15, 30 and 60 µg/mL, respectively. The fourth test tube without C12-50 was used as a control. All 4 tubes were vortexed to permit sufficient mixing and incubated at room temperature. After 1, 2 and 3 h, 10-1 dilutions in water were prepared for each tube, out of which 100 µl aliquots were taken and spread on TSA plates followed by incubation at 37 °C overnight.

Results and Discussion Models for interaction of antimicrobials with bacterial membranes Three models for the interaction of antimicrobial peptides with the bacterial membrane have been proposed.28 In the barrel-stave model, peptide helices form a bundle and insert into the membrane, like a barrel composed of helical peptides as the staves. In the toroidal pore model, antimicrobial peptide helices insert into the membrane, and induce the lipid monolayers to bend continuously and create the pore. In the carpet model, peptides accumulate on the membrane like a carpet. At high concentrations, surface-oriented peptides disrupt the bilayer in a detergent-like manner, eventually leading to the formation of micelles/vesicles.28 Model membrane studies have shown that most antimicrobial peptides follow the toroidal pore model or carpet model but not the barrel stave model. This is because the peptides in the barrel-stave model need to insert into the membrane without deformation of the lipid bilayer whereas peptides in the toroidal pore and carpet model tend to induce deformation (such as curvature) on cell membrane,40 which also 8 ACS Paragon Plus Environment

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can be observed in our AFM images as discussed below.49 While these three mechanisms are generally accepted, they are too rigid to explain all interactions taking place in a complex membrane.50 For example, mechanosensitive channels are involved in osmotic homeostasis serving as emergency release valves protecting cells from acute decreases in osmotic environment.51, 52, 53, 54, 55, 56 Disruption of mechanosensitive channel function due to stress caused by chemisorption of AMP polycations may play a role in rapid lysis.

Calculations of concentration based on theoretical predictions An extension of our prior polycation chemisorption (PCC) model5, 6 for C12-50 chemisorption and membrane disruption is shown in Figure 1A. This model is an adaptation of the carpet-like mechanism,28 which has been accepted to explain the activity of several antimicrobial peptides including dermaseptin S,21 melittin, magainin40 and other antimicrobial peptides. To address interactions of a synthetic amphiphilic polycation with a bacterium, we previously simulated C12-50 polycation chemisorption (PCC) on E. coli.6 The calculation asked “what if” complete chemisorption occurred, which may be thought of as surface coverage based on the carpet model.28 The dimensions of the selected copolyoxetane chain were estimated using the valence angle model.57 The resulting RMS end-to-end distance gave a “diameter” of 2 nm, which was an underestimate as side chains were not included. Also, distortions of the random coil conjugating with the bacterial membrane are unknown and were not taken into account. Using typical dimensions for E. coli surface area and hexagonal close packing, 2.6 ×106 polycation chains were estimated for bacterium coverage. The concentration in solution prior to complete chemisorption that corresponds to 2.6 ×106 polycation chains is 2 µg/mL. Considering the approximations for PCC model, it is interesting if not surprising that a calculation based on geometric considerations gives a concentration close to the minimum inhibitory concentration (MIC) for E. coli, strain Dh5α.6 For complete kill, the minimum biocidal concentration (MBC) is typically a factor of 2-3 higher than MIC.58 The results from the PCC model, which underestimates the MIC, are therefore consistent with a sorption – desorption equilibrium in solution and the notion that those polycations with optimum amphiphilic balance adsorb to the bacterial wall most strongly and 9 ACS Paragon Plus Environment

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effect maximum disruption at low concentrations. These considerations suggested exploring synthetic polycation membrane disruption in situ to better understand the relationship of chemisorption to pore formation and osmotic processes leading to cell membrane destabilization.

Biocidal effectiveness for copolyoxetane C12-50 Previously, we reported conventional determinations of MICs for copolyoxetane polycations with quaternary and PEG-like side chains.5 With a different strain of E. coli (Dh5α) than that employed herein, C12-50, 1 was one of the more effective copolyoxetanes with a MIC for (G-) E. coli of 7.3 (±0.6) µg/ml.6 A related copolyoxetane (C12-43) effected 2 log kill in 2 h for a concentration of 4 x MIC, ~30 µg/mL. These MIC determinations were made using a 24 h incubation time at 37 ºC. In the present study, AFM imaging employed an alternative E. coli strain ATCC #35218. As described in the Experimental Section, bacterial cells were immobilized on mica using a poly-Llysine fixation method.44 Growth medium was not employed for AFM imaging as preliminary studies showed that tip chemisorption resulted in image deterioration, but PBS was used to achieve a physiological pH (~7.4). This strategy also provides cells with water and essential ions to maintain a viable condition for short periods of time during treatment with C12-50 outside of the regular growth environment. Since the same cells are imaged over time, the overall AFM imaging time was short (1-3 h) to reduce the effect of thermal drift at the nanoscale. Given differences in incubation time in PBS (1-3 h versus 24 h), lack of growth medium and temperature (~25 versus 37 °C), a macroscale procedure was used to estimate the biocidal effectiveness of C12-50 for E. coli strain ATCC #35218 that paralleled nanoscale AFM imaging conditions. From this study at the 1x PBS concentration used for AFM imaging, 15 µg/mL C1250 effected a ~4 log reduction in 1 h (Figure 2). Higher concentrations (30 and 60 µg/mL) gave similar results. Despite the different conditions employed for this study, the results are consistent with previous work where the MIC of C12-50 for a different E. coli strain was 7.3 µg/mL.5

Real time AFM imaging of E. coli 10 ACS Paragon Plus Environment

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To observe the effect of the copolyoxetane polycation C12-50 on the Gram negative E. coli, AFM images were acquired before (0 h) and after adding C12-50 into the buffer solution (1, 2 or 3 h).. As a control after poly-lysine fixation, cells not exposed to copolyoxetane were imaged at the same time intervals in PBS. Control cell surfaces remained smooth with no detectable change for cell membranes even over 3 h (Figure 3A – 0H and 0A and 3H and 3A). In the figures, H represents the height image and A represents the amplitude image and the number refers to the time. Additional evidence that cells were unaffected by poly-lysine fixation and immersion in buffer was the negligible change in cell surface roughness over 3 h (Table S1, SI). C12-50 at 15µg/mL. Incubation of E. coli with 15 µg/mL after 1 h at ambient temperature resulted in major bacterial membrane disruption. A number of pores formed on the cell surface (~50 nm in diameter) two of which are marked by green arrows (Figure 3b-1H and 1A). Accompanying pore formation, bulges are seen part of which is marked by magenta arrows (Figure 3b-1H and 1A). Along with these, cell surface roughness increased from 5.6 ± 0.7 nm to 11.3 ± 2.1 nm over 3 h (Table 2, SI). Interestingly, after 3 h, no additional changes were observed compared to Figure 3B at 1 h (Figure S4, Supporting Information). The 50 nm pore size may be compared with a model for the mechanics of bulging of the cytoplasmic membrane through pores in the cell wall.59 This model resulted in the finding that the E. coli membrane undergoes a transition between a nearly flat state and a spherical bulge at a critical pore radius of ∼20 nm. We propose that the images shown in Figure 3b indicate a “state-of-the-surface” represented by Figure 1B. That is, C12-50 chemisorption is extensive at this concentration and time. Pores and bulges signal chemisorption and membrane damage (Figure 1B, green pore). While the macroscale bactericidal concentration study for C12-50 described above showed a 4 log kill at 15 µg/mL, a direct and unequivocal extrapolation of topological changes and pore formation to a single cell cannot be posited. Nevertheless, the dramatic changes from the control image to 1 h after introducing C12-50 are reasonably attributed to cell death. C12-50 at 30 µg/mL. Compared to images at 15 µg/mL, Figure 4 shows several cells with more extensive morphological changes after incubation with C12-50 at 30 µg/mL. As above, prior to addition cell surfaces are smooth and no damage to the cell outer membranes are observed


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indicating that cell morphology was unaffected after fixation and imaging (Figure 4-0H and 0A). As for 15 µg/mL, after 1 h incubation with C12-50 at 30 µg/mL, pores on cell surfaces were evident (green arrows, Figure 4-1H and 1A). However, the pores are larger crater-like nanofeatures (~100 nm) that are more numerous compared to images at 15 µg/mL. These features are attributed to the removal of LPS and outer membrane. After 2 h, bulges that form on the cell surface (marked by yellow arrows in Figure 4-2H and 2A) are more pronounced compared to the lower concentration. Interestingly, the largest bulges are observed on the same areas marked as pores at 1 h (Figure 4-3A). This implies that the C12-50 further interacts with the inner membrane after partial removal of the outer membrane resulting in bulging of the cell surface. There was no significant change in cell at 3 h compared to 2 h. The remarkable sequence shown in Figure 4 tracks the time dependent membrane disruption of C12-50. Imaging at 1 h correlates with high C12-50 chemisorption and membrane penetration (green indent) depicted in Figure 1B. These morphological changes lead to further disruption after 2 h in the form of bulges (Figure 4-2H and 2A, yellow arrows) represented by the magenta line in Figure 1B. The quantitative analyses across different individual cells and cultures show that these morphological changes are also associated with the increase of cell surface roughness (Table 3, SI). C12-50 at 60 µg/mL. At a higher concentration, after only 1 h incubation with 60 µg/mL, both vesicle-like features (marked by yellow arrows) and leakage of cytoplasmic fluid (marked by red arrows) were observed (Figure 5-1H and 1A), indicating damage to the inner membrane.40 The cell surface roughness also increased markedly in 1 h (~ 4 times), Table 4, SI. No further morphological change occurred between 1 and 3 h incubation, indicating cell death at 1 h. Leaking of soft and fluidic cytoplasmic content compromised the resolution of AFM images, which further reveals the dynamic process of antimicrobial activity at the single cell level. This also made the AFM imaging difficult once the cell had ruptured. Importantly, cytoplasmic content was flowing from the septal region of the cell. This observation may be connected to the presence of cardiolipin, a negatively charged phospholipid, which is generally located at the septal region of the E. coli inner membrane.60 Given the presence of cardiolipin, the leakage may be due to a conjugated product of cardiolipin and the polycation C12-50.


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Comparison of morphological changed by C12-50 with AMPs. Overall, AFM imaging showed that that the activity of the copolyoxetane C12-50 was time and concentration dependent. At 15 µg/mL C12-50 resulted in the appearance of nanoscale pores on cell surface, as well as the formation of bulges on the membrane surface. The outermost membrane disruption in the formation of pores and bulges are reasonably attributed to cell death as the accompanying biocidal concentration study showed 99.99% kill. Interestingly, at this lowest concentration investigated, the rigid peptidoglycan layer beneath the outer membrane maintains the cell structure and prevents cytoplasm leakage within the 3 h investigation. At 30 µg/mL, the changes seen at 15 µg/mL are amplified but again, the peptidoglycan layer prevents cytoplasm leakage. The enhanced membrane disruption is attributed to detergent-like effects after cell death. At 60 µg/ml C12-50 three stages of morphological change with time include pore formation, bulges and cytoplasm leakage. Initial chemisorption (< 15 µg/ml) corresponds to the carpet model (Figure 1A) that leads to membrane disruption (15 µg/ml) shown in Figure 1B. Further changes that result in cytoplasm leakage (60 µg/ml) are detergent like and occur after cell death. Pore formation, protrusion or bulges, increase of surface roughness and the leakage of cytoplasm are morphological changes induced by AMPs, but differences in media, concentration and AFM scanning conditions do not provide a direct comparison. For example, high speed AFM was used to image E. coli in water with AMP CM15 at 10-50 mg/mL (2 and 5 × MIC, respectively).44 Rapid formation of a corrugated surface was observed with some bacteria being affected at short times (~10 sec) and others at longer times (30 min). About 30% bacterial mortality was observed in 30 min for a control. Although E. coli adapts to a wide range of conditions61, 62 we are concerned about using water as a medium for imaging due to the osmotic-pressure differential across the cell membrane that might introduce change unrelated to C12-50.59 Unlike imaging using water as a medium, we do not observe surface features or E. coli cell death even after 24 h in 0.1 PBS (no C12-50) and cannot compare kill kinetics and morphological changes. Several AFM studies have investigated morphological changes induced by AMPs. Imaging was carried out in air after exposure of bacteria to the AMPs in solution. For example, Sushi peptides are AMPs that are comprised of 34 amino acids based on the sequence of two regions of “factor C” (Sushi 1 and Sushi 3).63 Sushi peptides have high bactericidal activity against Gram negative 13 ACS Paragon Plus Environment

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P. aeruginosa with 90% biocidal effectiveness at 0.06 - 0.25 µg/ml and low cytotoxic effects. After exposure of E. coli to Sushi peptide, AFM imaging in air showed micron scale indentations on cell surfaces.42 Using a similar imaging method, Alves investigated morphological effects of BP100, a short cecropin A-melittin hybrid peptide and pepR, an AMP derived from the dengue virus capsid protein. Images obtained in these studies show micron scale topological changes such as indentations and bulges but since AFM imaging was done in air, it is difficult to understand the exact origin of the micron-scale indentations. Further, interpretable nanoscale features are generally absent.

Effect on the surface hydrophobicity of E. coli: a connection to the carpet model The surface hydrophobicity of E. coli has been an important biomarker relevant to adhesion to inanimate surfaces and interaction with antimicrobial peptides. 64, 65 To study further the antimicrobial mechanism and especially the initial interaction with the cell membrane and effect on cell surface biochemical nature, AFM-based chemical force microscopy (CFM) was used to map the nanoscale hydrophobicity distribution on a single E. coli surface. AFM tips functionalized with undecanethiol present a -CH3 terminal group to the surface under interrogation. Using these functional AFM tips as probes, adhesion forces between the tipbound-CH3 groups and surface hydrophobic groups on bacteria can be determined. This technique enables mapping spatial distributions of hydrophobic groups and changes in hydrophobicity in real time.31, 66 Figure 6 shows the overlaid height and adhesion force maps of an E. coli incubated with C12-50 at the biocidal concentration of 30 µg/mL at t = 0, 1.5 and 3 h. Figure 5a indicates that the cell surface hydrophobicity was homogeneously distributed before addition of C12-50. The hydrophobicity of the E. coli surface originates from the cell surface components such as lipids and hydrophobic regions of membrane proteins.67 It is important to note that the map shown in Figure 5 represents a relative value of hydrophobicity across the cell surface. In terms of the adhesion force value, the E. coli surface hydrophobicity is lower than highly hydrophobic bacterial surfaces such as Aspergillus fumigates and Mycobacterium bovis.68 The change in cell surface hydrophobicity can therefore be used as an internal reference to monitor how the 14 ACS Paragon Plus Environment

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amphiphilic polycation affects the cell surface biochemical nature. After adding C12-50, the adhesion events on cell surfaces decreased precipitously at 1.5 and 3 h (Figure 6b, 6c). As a quantitative measure, the hydrophobicity of the single cell was calculated as a percentage of adhesion events to the total number of traces collected on the cell surface (Table 5-6, SI). The hydrophobicity of the E. coli at 1.5 and 3 h (~15%, Figure 7c) is much lower than E. coli at 0 h (~ 91%). As a control experiment, adhesion force maps were collected on E. coli in PBS at 0 and 3 h. Figure 7a and 7b shows that there is no obvious change after 3 h in terms of cell surface hydrophobicity. Overall, the hydrophobicity mapping on single cells shows that the entire cell surface hydrophobicity decreased extensively with the action of copolyoxetane. A model to explain the drastic difference in hydrophobicity is integrated into Figure 1. In this depiction of C12-50 chemisorption, interactions with negatively charged groups and lipid A on LPS result in quat groups (red side chains) preferentially occupying the E. coli interface. Hydrophilic PEG-like ME2 groups (blue side chains) are predominant at the water interface and account for the hydrophilic AFM image.64 This study provides a novel and independent metric demonstrating that the copolyoxetane initially binds to the cell outer membrane in a carpet-like fashion (Figure 1A). At 30 µg/mL C12-50, these results support the homogeneous polycation chemisorption (PCC) model for membrane disruption posited earlier.5, 6 A differentiating feature in adaptation of the carpet model for polycation C12-50 compared to polypeptide membrane disruption is a fundamental difference in chain structure. The carpet model is based on a helical structure for polypeptides. For example, Brogden’s depiction of the carpet model shows helical peptide chains adsorbing to the membrane by orienting parallel to the surface of the lipid bilayer and forming an extensive layer.28 The chain architecture and physical properties of C12-50 are completely different. C12-50 is a viscous, colorless liquid that is ~ 45 °C above the glass transition temperature at ambient conditions. Other than Tg, which is characteristic of polyoxetanes,69, 70 no thermal transition due to side chain association or other associative phenomenon is observed at higher temperatures. While soluble in water, the propensity to undergo association in water to form aggregates such as micelle formation at concentrations in the vicinity of the MIC or bactericidal concentration has not been fully explored. However, we suspect that self-association, which occurs for polypeptides and plays a 15 ACS Paragon Plus Environment

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role in membrane disruption, may be disfavored for copolyoxetanes due chain flexibility and copolymer structure. Consequently, the model in Figure 1 assumes C12-50 is a random coil that chemisorbs to the bacterial outer membrane. The driving force for this association is similar to that for polypeptides, namely ionic forces, but the nanoscale model is completely different.

Conclusion

In this study, real-time atomic force microscopy was used for analyzing the antimicrobial effect of copolyoxetane P[(C12)-(ME2Ox)-50/50], C12-50, 1, which is a member of a novel class of synthetic amphiphilic polycations (Scheme 1). Interactions of C12-50 with the membrane of a model E. coli bacterium were studied by imaging in real time, in a fluid environment and at physiological pH. AFM images show cell membrane changes with increasing C12-50 concentration and time including nanopore formation and bulges associated with outer bacterial membrane disruption and cell death. The sequence of processes for initial membrane disruption by the synthetic polycation C12-50 follows the carpet model posited for antimicrobial peptides (AMPs). However, the nanoscale details are distinctly different as C12-50 is a synthetic, water soluble copolycation that is best modeled as a random coil. By this adaptation, C12-50 at 15 µg/mL initially leads to a “carpet” of chemisorbed copolyoxetane polycations and formation of pores and bulges. At the highest concentration (60 µg/mL) further cell membrane disruption results in leakage of cytoplasm driven by detergent-like action. Chemical force microscopy shows that incubating cells with C12-50 decreased the hydrophobicity across the entire cell surface at an early stage, provides evidence that C12-50 polycations initially bind with cell membrane in a carpet-like fashion. In fact, the hydrophobic functionalized tip appears to be a perfect foil for the polycation antimicrobial which appears to concentrate hydrophilic PEG-like groups (blue, Figure 1) at the polymer coil / water interface while the quat side chains are enthalpically driven to the E. coli membrane via ionic interactions (red, Figure 1). Future work is planned to explore this important finding. Real time AFM imaging described herein elucidates the mechanism of antimicrobial action for 16 ACS Paragon Plus Environment

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polycation C12-50 chemisorption and membrane disruption at the single cell level. This new and detailed imaging opens the door to detailed studies for identifying morphological changes that define the minimum inhibitory concentration and the minimum biocidal concentration for polycation antimicrobials. These fundamental studies on polycations are expected to shed light on why seemingly small changes in chain structure result in radical changes in antimicrobial activity.71 Taken together, the real time AFM approach elucidates the mechanism of antimicrobial action for copolyoxetane C12-50 at the single cell level and provides a pathway to insights into mechanism of action of other synthetic amphiphilic polycations.

ACKNOWLEDGEMENTS KJW thanks the School of Engineering Foundation for partial support of this research. KJW also acknowledges partial support from the National Science Foundation, Division of Materials Research, Polymers Program (DMR-1206259). VKY and CEJ thank the VCU Presidential Research Quest Fund for partial support of this research. Other support was obtained by DEO from the NIH (R01 AI19146) and Veterans Administration Medical Research Funds (I01 BX000477)


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Br

NaOH TBAB

OH +

Br

Br

O

O

Water/Hexane

Ox

O

BBOx

OMe O

Br O

O

NaH +

HO

OMe

O

O

THF

BrOx

ME2Ox

OMe

Br

OMe

Br

O O O

O

BF3/BD

+

DCM

O

BBOx

O

O

O

ME2Ox

H

OMe

ME2

Br N

O

m

CH3 (CH2)10

OH

O

O

n

O

O H

m

C12

CH3(CH2)11N(CH3)2

CH3CN

OH

O

O

n

Scheme 1.Synthesis of copolyoxetane, P[C12-ME2Ox)-m/n], shown with quat “C12” and PEGlike “ME2” side chains.


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Figure 1. Depiction of C12-50 chemisorption on the E. coli membrane: A, chemisorbed C12-50 is shown as a random coil with C12 side chains (red, at membrane interface) and ME2 side chains (blue, at water interface); B, pore (green outline) and bulge (magenta outline) are colorcoded to match AFM features denoted by colored arrows; drawing is not to scale as the idealized C12-50 chain has a root mean square end to end distance of ~2 nm.


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Figure 2. Representative E. coli agar plates after 1 h incubation: control and with C12-50, 15 µg/ml. Approximately 4 log reduction in bacterial colonies (99.99% kill) was observed.


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Figure 3. AFM imaging of E. coli: H = height; A = amplitude at 0, 1 or 3 h. (a) E. coli in PBS (control); (b) E. coli in PBS with C12-50 = 15 µg/mL; green arrows mark two of several pores; magenta arrows mark bulges. Scale bars: 500 nm.


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Figure 4. Height (H) and amplitude (A) images for E. coli for 1, 2 and 3 h at C12-50 = 30 µg/mL. Green arrows mark some of the pores observed on cell surface. Magenta arrows indicate selected bulges on the cell surface. Zero time height and amplitude images (0H and 0A) are obtained before addition of C12-50. Scale bars: 500 nm.


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Figure 5. Effect of concentration of C12-50, 60 µg/ml, on E. coli at t = 0 and 1 h. Yellow arrows: bulge features. Red arrows: leaking cytoplasmic content. Scale bars: 500 nm.


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Figure 6. Observation of surface hydrophobicity of the E. coli cell surfaces before (a, 0 h) and after adding 30 µg/ml of copolyoxetane (b, 1.5 h and c, 3 h). Scale bars: 300 nm. The force map is overlaid on the topography map to obtain this representation. The color of pixels indicates the magnitude of force value.


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Figure 7. Control experiment: Overlay of force and height profiles for the hydrophobicity on E. coli cell surfaces in PBS (no antimicrobial). (a) 0 h. (b) 3 h. Scale bars: 200 nm. (c) Analysis of binding % shows the hydrophobicity of single E. coli cells decreased after adding copolyoxetane. The surface hydrophobicity does not change in PBS in 3 hours. The data are from three independent experiments for each system.


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