Living Bacteria-Nanoparticle Hybrids Mediated through Surface

Subscriber access provided by UNIV OF SCIENCES PHILADELPHIA

Biological and Environmental Phenomena at the Interface

Living Bacteria-Nanoparticle Hybrids Mediated through Surface-displayed Peptides Hong Dong, Deborah A Sarkes, Jeffrey J Rice, Margaret M. Hurley, Adele Fu, and Dimitra N. Stratis-Cullum Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b00114 • Publication Date (Web): 25 Apr 2018 Downloaded from http://pubs.acs.org on April 25, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Cover Art 214x279mm (300 x 300 DPI)

ACS Paragon Plus Environment

Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 36

Living Bacteria-Nanoparticle Hybrids Mediated through Surface-displayed Peptides

Hong Dong †,‡,# , Deborah A. Sarkes†,#, Jeffrey J. Rice§⊥, Margaret M. Hurley†, Adele J. Fu†⊥, Dimitra N. Stratis-Cullum*,†

†

US Army Research Laboratory, Biotechnology Branch, 2800 Powder Mill Road, Adelphi, MD 20783 ‡

General Technical Services, 1451 Route 34 South, Wall, NJ 07727

§

Department of Chemical Engineering, 212 Ross Hall, Auburn University, Auburn, Alabama 36849 ⊥Oak

Ridge Associated Universities, 4692 Millennium Drive, Suite 101, Belcamp, MD 21017

*Corresponding Author: [email protected] #Author

Contributions: H. Dong and D. Sarkes contributed equally to this work.

Keywords: living bacterial hybrids, cell viability, surface-displayed peptides, assembly, metal nanoparticles, gold binding peptides.

1|P a g e


Page 3 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

ABSTRACT In this study, we investigated the preparation of living bacteria-nanoparticle hybrids mediated by surface-displayed peptides. The assembly of metallic nanoparticles on living bacteria has been achieved under mild conditions utilizing metal-peptide interactions, while the viability of the bacterial cells were greatly preserved. Escherichia coli was engineered with inducible gene circuits to control display of peptides with desired sequences. Several designed peptide sequences as well as gold binding peptides were expressed on the cell surface using enhanced circularly permuted outer membrane protein X (eCPX) scaffolds. Driven by metal-peptide affinity, “bio-friendly” citrate-stabilized gold nanoparticles were self-assembled onto the surface of bacteria with displayed peptides, which required overcoming the repulsive force between negatively charged nanoparticles and negatively charged cells. The bacteria/Au NP hybrids were highly viable and maintained the ability to grow and divide, which is a crucial step towards the creation of living material systems. Further activity and preservation of the bacterial hybrid assembly was demonstrated. The method described herein enables the conjugation of bacterial surfaces with diverse metal-rich nanoparticles in an inducible, and therefore easily controlled, manner. The expressed peptide sequences can be easily modified to alter binding affinity and specificity for a wide variety of materials to form on-demand, high density living biohybrids.

2|P a g e


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 36

INTRODUCTION Integration of living bacterial cells with inorganic components to create living functional hybrids has the potential to dramatically change how materials are used and to enable novel functions derived from the combination of biology and materials science.1 Living cells interfaced with metallic nanoparticles and other nanomaterials can perform functions often unique from their origins,2 or demonstrate improved tolerance against harsh environments due to the formation of a protective nanoshell3-4. Due to these improved properties, living cell/nanoparticle hybrids have found a wide range of applications, including bio-supported metal catalysts, biosensors, bioelectronics and bio-imaging.2, 5-8 9-10 A variety of methods have been reported for loading metallic nanoparticles onto bacterial cells. The simplest chemical methods take advantage of the overall negative charge of E. coli,11 utilizing

electrostatic

interactions

to

deposit

cationic

nanoparticles,

such

as

cetyltrimethylammonium bromide (CTAB)-stabilized gold nanoparticles,6 onto negatively charged bacterial surfaces. Although success in maintenance of cell viability has been achieved for the gram-positive species Bacillus cereus after deposition of poly(L-lysine)-coated 30-nm Au NPs,10 it is known that nanoparticles functionalized with cationic side chains are moderately toxic to gram-negative E. coli as a result of their interactions with the cell membrane.12 An alternative method is to synthesize gold nanoparticles in situ by introducing the cells to a metal salt solution with or without the addition of a reducing agent.5, 13-14 While this technique has been proven to produce coatings up to ~8 nm thickness on E. coli,14 cell viability in these studies was reported but not quantified. The nanoparticle synthesis process using metal cations and other toxic reagents may affect the normal cell cycle. Preserving the viability of bacteria after functionalization with gold nanoparticles, as well as other types of nanoparticles, is of significant interest for many 3|P a g e


Page 5 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

applications. Using ‘‘biofriendly’’ approaches allows for the functionalization of intact living cells with metal-rich nanoparticles while avoiding potentially cytotoxic effects on bacterial cells. Other studies have attempted to address this by using a layer-by-layer (LbL) processes in which a protective positively-charged polymer coat is placed on the bacterial cell wall before exposure to citrate-reduced Au NPs. Kahraman et al.15 successfully demonstrated this technique on E. coli using roughly 13 nm size Au NPs. The distribution of nanoparticles on the bacterial surface in this work was shown to be non-uniform, and cell viability was not tested, as the intended application was surface-enhanced Raman scattering (SERS) for single bacterial cell identification and hence did not have a viability requirement. Multiple groups have followed a different path and studied nanoparticle attachment at bacterial fibrils. These methods have the advantage of distancing the inorganic moiety from the cell wall, thereby avoiding potentially toxic interactions. Vedantum et al.16 exploited the natural mannose-binding properties of E. coli fimbriae to bind bacteria to functionalized 200 nm Au NPs and test the sugar specificity of various strains. Chen et al.17 interfaced E. coli curli fibrils to Au NPs via histidine chemistry to produce conductive switchable biofilms. Chaudhary et al.18 also harnessed carbohydrate-protein interactions to study glycol-Au NP adhesion to E. coli, also allowing variations in nanoparticle shape. This work provides an alternative approach to production of viable bacterial cell/nanoparticle hybrids suitable for sensing, catalysis, and delivery applications. It utilizes the highly promising and facile strategy of engineering peptides displayed on the cell surface to integrate functional nanomaterials directly with cells, with enhanced specificity. To achieve this, we have adopted an inducible bacterial display system that has been used repeatedly to identify high affinity peptides towards various protein targets.19-26 The enhanced circularly permutated OmpX (eCPX) scaffold used here is an engineered variant of the naturally-occurring bacterial transmembrane protein 4|P a g e


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 36

“outer membrane protein X” (OmpX) that has undergone directed evolution to improve peptide display.19-21 Unlike OmpX, eCPX presents both C- and N-termini to the exterior of the cell.21 This enhanced biterminal display scaffold allows diverse peptides to be fused to both termini and displayed on the cell surface upon induction. By engineering the scaffold at the DNA level, cells with specific peptides displayed have been shown to selectively adhere to different inorganic substrates27-28 or modified surfaces.29 Taking advantage of this bacterial expression system, which produces thousands of copies29-30 of the eCPX protein scaffold per cell with appended engineered peptide sequences, desired functionalities at the cell surface can be achieved via binding of surface peptides with nanomaterials or molecules (e.g. fluorescent probes), using physical interactions or chemical bonding. Furthermore, in contrast to methods relying on broad-brush electrostatics and manipulation of the Au NP surface chemistry, the eCPX scaffold is under the control of an inducible promoter and utilization of this functionality can be delayed until the desired time. Moreover, the ability to engineer bacteria to express desired peptide sequences enables bioconjugation of various nanoparticles and functional molecules to the cargo-carrying bacteria by tuning peptide-cargo interactions. The method demonstrated herein is therefore extendable to a wide variety of materials and applications. We postulate that metal-peptide affinity could drive assembly of pre-formed metallic nanoparticles to the surface of bacterial cells, which are genetically engineered to display specific peptide sequences. Peptides that selectively bind to metals with high affinity, such as gold-binding peptides, have been identified using phage and cell-surface display technologies.31-33 Peptide sequences chosen for this work range from traditional gold binding sequences (GBP1 and GBP2) to engineered sequences that probe the interplay between sidechain chemistry and structure in the binding process. The latter sequences utilize histidine and methionine, which are known to have 5|P a g e


Page 7 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

an affinity towards gold.34-35 We utilize the novel “methionine tag” of Okada et al36 who investigated the relative binding efficiency of methionine, cysteine, and glycine-containing sequences to gold magnetic particles. Histidine based sequences are studied to mimic the chemistry of polyhistidine-appended proteins which are known to self-assemble onto gold nanoparticles.37 The P2X peptide sequence expressed at the C-terminus of the eCPX scaffold for fluorescence analysis is also shown to have gold-binding characteristics, and its role in cell adhesion is explored. In this study, we investigate affinity-driven assembly of metallic nanoparticles onto living bacterial cells under mild conditions, utilizing genetic engineering and bacterial expression systems for peptide display. The peptides were genetically fused to the N-terminus of eCPX and expressed in an MC1061 strain of E. coli. The effects of both peptide sequence and gold nanoparticle size were studied and compared for their ability to promote stable peptidenanoparticle interactions. Nanoparticle coverage and aggregation on the bacterial cell is quantified using protocols developed from the literature.18, 38 Notably, the bacterial cells immobilized with citrate-stabilized gold nanoparticles maintain high viability, which is important for applications that require living cells. Our study demonstrates the creation of living hybrid systems in a controlled inducible manner, utilizing the tools of both biology and materials science.

EXPERIMENTAL METHODS Cloning of E. coli Expression Plasmids The eCPX 3.0 bacterial display peptide library was obtained through a collaboration with Patrick Daugherty and Cytomx. An empty pBad33-eCPX clone was isolated from the library for use as a negative control and was modified for cloning purposes to replace the SfiI site before peptide

insertion

(GGCCAGTCTGGCC)

with

an

AatII

site

(GACGTC

within

6|P a g e


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 36

GGGACGTCTGGCC, for minimal disruption to translated sequence: a single Q to T mutation) just before the region of random peptide insertion at the N-terminus of the mature scaffold, creating the “pDSJR” plasmid. This minimal mutation to the AatII restriction site allows for easy cloning of inserts already incorporated into other versions of the eCPX plasmid into this new version using PCR. Mini genes were ordered from Bio Basic for this purpose and included the upstream KpnI site and downstream XhoI site. Cloning was performed by digesting the mini genes and the pBad33-eCPX plasmid with KpnI-HF and XhoI overnight at 37°C and ligating appropriate fragments using T4 DNA ligase for 1 hour at room temperature. The restriction site mutation was confirmed by DNA sequencing (Genewiz). The plasmid was further altered to express specific peptides (see Table 1) at the N-terminus of eCPX and to remove the P2X peptide from the Cterminus of eCPX using standard cloning techniques. Details of creating “pDSJR-No P2X” plasmid and cloning of the various gold binding peptides into the pDSJR and pDSJR-No P2X plasmids are provided in the supporting information. Assembly of Au Nanoparticles with cells The bacterial strains were grown in LB medium with 25 µg/mL chloramphenicol (LBcm25) at 37°C, shaking at 225 rpm. After the optical density at 600 nm (OD600) reached about 0.5, the cells were induced to express the eCPX display scaffold by adding 0.04% w/v L-arabinose (diluted from a 4% w/v aqueous solution). The cell cultures continued to incubate at 37 °C with shaking at 225 rpm for 1 hour. The induced cells were then centrifuged at 5000 rpm for 5-10 min and the supernatant was discarded. The bacteria were washed with PBS (pH 7.4) by centrifugation and resuspension of the cell pellet. Finally, the bacteria were reconstituted in PBS and their OD600 was measured. To prepare filamented bacteria, the cells were grown using similar procedures with the addition of 10 µg/mL aztreonam to the grown medium (for suppression of cell division). The

7|P a g e


Page 9 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

filamented cells were induced at OD600 of 0.5 with 0.04% w/v L-arabinose and incubated at 37 °C with shaking for 1 hour before harvesting. Citrate-stabilized gold nanoparticles (Au NPs) with diameters of 10 and 20 nm were obtained from Ted Pella and NANOCS, respectively. For loading Au NPs onto the cells, the solutions of Au NPs and cells in PBS were combined at a v/v ratio of 1:9 Au NPs to cells. Briefly, the cell cultures in PBS were diluted to achieve an optical cell density (OD600) of 0.28 in the mixture. The cells were then incubated with Au NP solution (100 µL/mL) for 1 hour with gentle agitation before analysis. Evaluation of live/dead cell ratio A live/dead staining assay was performed using the BacLight Bacterial Viability L7012 kit to examine viability of cells after loading nanoparticles. Upon completion of the experimental treatments described above, a staining solution containing a 1:1 mixture of SYTO 9 (3.34 mM) and propidium iodide (20 mM) was added to the sample solutions (1:500 dilution). After incubation at room temperature for 15 min in the dark, 20 µL of each sample was analyzed using a glass slide and cover slip. The samples were examined by fluorescence microscopy (Nikon Eclipse TE2000-E) using a 40× objective, and images of randomly chosen fields of view were captured. The relative number of live (green) versus dead (red) bacteria for each sample was analyzed for at least five images using ImageJ (National Institutes of Health) to obtain the average numbers of live and dead cells. Optical interference of Au NPs alone was also assessed by fluorescence microscopy with no bleed through signal observed in either channel. Flow cytometry was used to quantify the viability of cells bound to Au NPs. The samples were stained with propidium iodide (1:500 dilution) in the dark for 15 min, followed by analysis of red 8|P a g e


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 36

fluorescence intensity using fluorescence-activated cell sorting (FACS) to differentiate dead cells (stained) and live cells (unstained) for 25 µl of cell sample resuspended in 500 µl FACSFlow solution (BD). Unstained cells were run as a negative control and this parent population was gated on a scatterplot of FSC-A vs SSC-A to select cells and then gated again on a dependent PE-A vs FSC-A plot to account for autofluorescence. Cells with and without 10 nm or 20 nm Au NPs were compared. For each sample, 10,000 events were recorded to determine the percent viability (percent remaining inside of the gate on the PE-A vs FSC-A plot; percent falling outside of the gate have compromised membranes) and the normalized median fluorescence intensity (nMFI), further described in supporting information for similar FACS experiments. The percent viability reported has been normalized to the percent viability of stained cells without Au NPs. As a further test of cell viability, a regrowth assay was performed. Bacterial cultures with and without bound Au NPs were diluted to the same OD600 in fresh LBcm25 medium, followed by shaking incubation at 225 rpm and 37°C for 3.5 hrs. Optical density measurements at 600 nm were taken for each sample at 15-30 min intervals to create a growth curve for each and confirm that that the Au NPs did not negatively impact cell division. Viability and regrowth was further confirmed by overnight growth of 50 µl of sample on LBcm25 agar plates overnight at 37°C and 10 µl of sample in 5 ml liquid cultures overnight at 37°C, 225 rpm. TEM imaging and analysis The bacterial samples for transmission electron microscopy (TEM) analysis were prepared using the drop-casting method. The copper grids covered with holey carbon films were surfacetreated to become hydrophilic using UV-Ozone. A 20 µL volume of E. coli/Au NPs in PBS buffer was dropped onto the copper grid. After 3 min, the majority of the solution was removed by touching the edge of a piece of filter paper to the droplet. Then the grid was washed quickly with 9|P a g e


Page 11 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

drops of water two times, and air dried. The bacteria were not fixed prior to TEM imaging. The samples were viewed with a JEOL 2100F transmission electron microscope operated at 200 kV. Images were then analyzed using the Fiji distribution39 of the ImageJ40 image processing program. Similar to previous studies in the literature,

18, 38

image processing followed a protocol

outlined in Figure S1, in which an ellipse was fit to each individual cell, the background was subtracted, the threshold adjusted using the default algorithm and settings to ensure all nanoparticles were captured, the image was converted to binary, and particle analysis was then performed using default settings for particle size and circularity. Nanoparticle aggregate area data was accumulated from the particle analysis, and statistical analysis of the aggregate area distribution was performed using the NumPy41 and SciPy libraries42 in python 2.7. Quantities of interest calculated included area of coverage by nanoparticle aggregates as a fraction of the theoretical 2D surface area of each cell, aggregate area mean, mode and maximum, as well as skewness of the aggregate area distribution (Table S1). These use of aggregate coverage as a fraction of 2D surface area was chosen as a best representation of Au NP adhesion to the cell surface free from the effects of cell size and nanoparticle aggregation. Properties such as aggregate area mean and skewness (which provides a measure of asymmetry in the aggregate distribution profile and in this case provides an indication of shift to larger aggregate size) are provided to assess the degree of Au NP aggregation which is shown to vary greatly with peptide binding sequence. Slight variations from elliptical morphology can be seen due to the effects of the holey carbon TEM support and are expected from this methodology. Only individual (non-overlapping) elliptical cells were chosen for analysis. Error bars represent averaging over multiple cells, with a sample size ranging from N=2 (for the negative control) to N=6. This is also provided in the supplemental information (Table S1). 10 | P a g e


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 36

RESULTS AND DISCUSSION

Figure 1. (a) Binding of metal nanoparticles to surface-displayed peptides. (b) Bacterial outer membrane with eCPX display scaffold inserted (blue oval). The N-terminus of eCPX has been modified to display a specific peptide of interest after the N-terminal GTSGQ sequence. P2X peptide is displayed at the C-terminus for assessment of expression level. (c) Cells without inserted peptide sequence are used as a negative control but display all other features of the system. (d) eCPX scaffold displaying peptide at N-terminus without C-terminal P2X peptide, which was prepared as a second system to remove contributions of the P2X expression tag in the binding interactions. A peptide-free negative control (NC) is also available without P2X (pDSJR-No P2X NC).

Our goal was to prepare biohybrid living systems using the directed assembly approach illustrated in Figure 1a. E. coli were genetically engineered to display peptides with desired amino acid sequences on their outer membranes through inducible expression of the eCPX bacterial display scaffold. Direct interactions between these surface-displayed peptides and Au NPs were exploited to load functional nanomaterials onto the bacterial surface. Affinity was derived from the coordination or other bonding between peptides appended to the N-terminus of the membrane-

11 | P a g e


Page 13 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

embedded eCPX scaffold and the metal surface itself, rather than through ligands incorporated onto the metallic nanoparticles. Design and display of surface peptides Binding affinity of an individual amino acid residue in a peptide sequence is governed by its reactive side group as well as the peptide’s ability to access conformations favorable for adsorption to a material, such as gold.34 We designed two peptide sequences containing methionine residues (M6G9) or histidine residues (H6G9) (Table 1), as these residues exhibit strong binding interactions with gold.34-36 In accordance with previous studies delineating the importance of local flexibility in gold binding,43-44 glycine was used as a flexible spacer between our putative binding residues (M or H) to allow the peptide to adapt easily to conformations favorable for binding to gold. Two peptide sequences that have demonstrated strong binding to gold in prior studies, GBP1 (MHGKTQATSGTIQS) and GBP2 (ALVPTAHRLDGNMH), were also included in this study (Table 1). Au-binding peptides GBP1 and GBP2 were discovered using a cell surface-displayed combinatorial peptide library.31 Previous studies have demonstrated how the labile nature of GBP1 plays a role in gold binding.45 These peptide sequences were fused to the N-terminus of the eCPX protein using the same cloning strategy as the designed peptides, which inserts each peptide after the N-terminal sequence GTSGQ, as shown in Figure 1b. Cells without the insertion of gold binding peptides were used as negative control (NC), on which only peptide segment GTSGQ was displayed at the N-terminus (Figure 1c). For reliable comparison of peptide binding to gold, it is necessary to ensure that the peptides are properly displayed on the cell surface. The presence of P2X, a fixed peptide fused to the Cterminus of eCPX (as shown in Figure 1b-c) with the sequence HISQWKPKVPNREDKYKK (Table 1), is a useful feature of the eCPX 3.0 display scaffold that can be harnessed for this 12 | P a g e


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 36

purpose. The P2X peptide promotes binding to a fluorescent fusion protein, YPet-Mona. Since it is present at the C-terminus, binding to YPet Mona is therefore only possible if the entire eCPX scaffold from N- to C-terminus is expressed and shuttled to the cell membrane. The display of P2X therefore enables quantitative affinity measurements relative to the overall expression level of eCPX and the peptide of interest. This is a critical step in understanding binding because some peptide sequences can cause poor expression or completely disrupt expression or shuttling of the eCPX scaffold to the cell membrane, which would cause a false negative result. Since the P2X peptide may additionally influence the ability of the cells to bind to metal, a separate cell system without expression of P2X was also prepared, enabling investigation of binding affinity with contributions solely from the investigated peptide sequences (Figure 1d). There is therefore a need to assess both with P2X and P2X-free versions of the scaffold, with and without N-terminal peptide. Taking advantage of the interaction between the C-terminal P2X peptide with the fluorescent protein YPet-Mona, flow cytometry experiments were carried out to verify that peptide display was successful. From the scatter plots derived from fluorescence activated cell sorting (FACS, supporting information Figure S2) we conclude that over 95% of the bacterial population was fluorescently labeled for each peptide listed in Table 1, which indicates that the peptides were properly displayed on most of the cells. For the eCPX system, the number of displayed peptides has been previously estimated to be on the order 103-104 copies per cell on the surface of E. coli.2930

13 | P a g e


Page 15 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Table 1. Sequences of peptides displayed at the surface of E. coli in this work Variants Negative Control (NC)

Peptide Sequences GTSGQ (Only)a)

P2X HISQWKPKVPNREDKYKK M6G9 MMMGGGMGGGMGGGM H6G9 HHHGGGHGGGHGGGH GBP1 MHGKTQATSGTIQS GBP2 ALVPTAHRLDGNMH a) All variants contain GTSGQ at the N-terminus before peptide insertion

Assembly of Gold NPs on Cells The assembly of gold NPs on bacterial cells was achieved by simply mixing the nanoparticle solution with the bacterial cells in phosphate-buffered saline (PBS). To visualize attachment of Au NPs on the bacteria, transmission electron microscopy (TEM) images were acquired on the bacterial hybrids. Negative control and peptide-displaying cells which also displayed the P2X tag at the C-terminus of eCPX (for expression confirmation) were first assessed. Figure 2a-b shows TEM images of cells with the genetic machinery encoding M6G9 with P2X (noted as M6G9-P2X) that have been combined with 20 nm, citrate-stabilized Au NPs with and without prior induction of the surface-displayed peptide. Cells without induction of peptides carry very few Au NPs (Figure 2a). In contrast, the cell surfaces were heavily populated with 20 nm Au NPs in the presence of surface-displayed M6G9-P2X peptides (Figure 2b). Therefore, the same cell line can be altered from a non-binding (un-induced) to binding (induced) form at the desired time through induction and expression of the Au-binding peptides. Slight differences in the darkness of the cells in Figures 2a and 2b are a result of differences in autocontrast in the presence of and absence of significant binding to Au NPs in these unaltered images. It is known that the outer membrane of E. coli is heavily populated with lipopolysaccharides and that the cell surface is negatively charged due primarily to phosphate groups.11 Unlike bare 14 | P a g e


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 36

gold surfaces, Au NPs and many other types of NPs have ligand-functionalized surfaces, which act to stabilize the NPs and prevent aggregation of NPs in solution. The Au NPs used in our study are citrate-stabilized and negatively charged. In the absence of surface binding peptide, the repulsive force between the negatively charged cell surface and the negatively charged Au NPs prevents effective attachment of NPs to the cells (Figure 2c). Due to the strong Au-methionine coordination,36 methionine from M6G9 peptide can partially displace the weak-binding ligand (the citrate molecules) on the Au NPs, which reduces or overcomes the charge repulsion force, resulting in attachment of citrate-stabilized nanoparticle to the negatively charged cell surfaces (Figure 2d). Figure 1a and Figure 2d illustrate how binding to Au NPs might look for a single peptide on the cell surface. This is possible, especially when the nanoparticle size is smaller than the distance between membrane proteins. Based upon preliminary investigations quantifying expression level of the similar (but unenhanced) circularly permutated OmpX (CPX) scaffold on the cell surface,46 we expect the N-terminal displayed peptides to have a broad distribution of spacing, which is dependent on the display level of the peptide. Expression analysis using flow cytometry shows that the display level of each peptide is very similar within each cell population for the peptides assessed here, with nMFI close to 1.0 (Figure S2), but the exact number of copies of displayed peptides were not analyzed. Because it is feasible that the eCPX scaffolds on the cell surface could be distributed at distances that are similar to the sizes of the Au NPs used in this work,46 and also due to the fluid nature of cell membranes,47 more than one peptide may bind to a single nanoparticle. Notably, many Au NPs were observed at the light-contrast edge surrounding the cells in the TEM images of the induced cells, confirming attachment of Au NPs to the surface of the outer cell membrane.

15 | P a g e


Page 17 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

a

c

b

d

Figure 2. (a-b) TEM images of bacterial strains bound to 20 nm Au NPs: (a) M6G9-P2X cells containing plasmid but without induction of peptide display (Un-induced: U-M6G9-P2X), (b) M6G9-P2X cells with induced N-terminal M6G9 and C-terminal P2X peptides. Statistical data shown in Figure 5a and Table S1. (c-d) Schematic showing replusion between negatively-charged bacterial cell membrane and negatively-charged Au NPs in the absence of binding peptides (c) versus overcoming this repulsive force by bacterial binding to Au NPs via surface-displayed peptides (d).

Figure 3a-d shows the attachment of 20 nm Au NPs to the negative control (which displays no N-terminal peptide but displays C-terminal P2X, noted as NC-P2X) and several other gold binding peptides, including H6G9, GBP1 and GBP2, which were displayed on eCPX with co-expression of the C-terminal P2X peptide (noted as Peptide-P2X). The presence of these peptides (H6G9P2X, GBP1-P2X, and GBP2-P2X) on the cell surface apparently drives populated nanoparticles to the cell surface, to a similar extent as observed for cells displaying M6G9-P2X (Figure 2b). It is also apparent from the representative images shown in Figure 3 that there is a difference in the number of loaded nanoparticles bound to the NC-P2X negative control (Figure 3d) as compared to the number bound to cells displaying M6G9-P2X and all other peptides tested thus far (Figure 2b, 3a-c), indicating that the inserted peptides function to increase affinity for gold, as expected.

16 | P a g e


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a

b

c

d

Page 18 of 36

Figure 3. TEM images showing that bacterial cells, with various N-terminal displayed peptides and fixed C-terminal displayed P2X peptide, bound to 20 nm Au NPs: (a) H6G9-P2X, (b) GBP1P2X, (c) GBP2-P2X, (d) NC-P2X. Statistical data shown in Figure 5a and Table S1.

Notably, NC-P2X cells also carry some Au NPs (Figure 3d), unlike the un-induced cells shown in Figure 2a. As mentioned, NC-P2X cells display the P2X peptide at the C-terminus in addition to the peptide segment GTSGQ at the N-terminus. This indicates that the P2X peptide, designed for quantification of expression, has some affinity for gold and contributes to the attachment of Au NPs

on

the

NC-P2X

cells.

This

is

not

surprising

since

P2X,

with

sequence

HISQWKPKVPNREDKYKK, contains a variety of putative gold-binding residues including histidine. Co-expression of two distinct peptides at each of the N- and C-termini is an attribute that could potentially be exploited to build more complex bacterial networks. To understand if P2X increases binding affinity when co-expressed with the peptides of interest, a second system was created to exclude the C-terminal P2X peptide from the eCPX scaffold. This pDSJR No P2X system displays the same gold binding peptides of interest at the N-terminus (after the GTSGQ sequence) but not the P2X peptide at the C-terminus (Figure 1c). These non-P2X variants (named 17 | P a g e


Page 19 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

herein using the peptide name only) were evaluated for binding to 20 nm Au NPs using the same procedures. It is important to note that expression levels of each peptide in both pDSJR and pDSJR-No P2X systems are expected to behave similarly since the same promoter and other regulatory elements are used in each case and the same batch of competent E. coli was for transformed with each of these plasmids. Figure 4a shows that very few Au NPs were attached to induced NC cells without P2X (noted as NC). The attachment of 20 nm Au NPs to NC cells is much lower in comparison to the attachment to NC-P2X cells, confirming that the P2X peptide, rather than the base structure of the eCPX scaffold itself, has binding affinity for Au NPs. Interestingly, M6G9 and H6G9 cells without display of P2X (noted as M6G9 and H6G9) (Figure 4) have similar levels of Au NP association as these same peptides with P2X (Figures 2 and 3). This suggests that the elimination of P2X does not significantly influence binding efficiency of 20 nm Au NPs to these cells, and that the gold binding peptides alone at the N-terminus can modulate high affinity binding to Au NPs. Comparison of Figures 4b and 4c with Figure 3d indicates that these engineered gold binding peptides have a stronger binding affinity to Au NPs than does P2X. In contrast, the number of 20 nm Au NPs bound on GBP1 no P2X (noted as GBP1) and GBP2 no P2X cells (noted as GBP2) (Figure 4d-e) were greatly reduced with P2X removal. This indicates that GBP1 and GBP2 peptides are relatively weaker binders for 20 nm Au NPs as compared to M6G9 and H6G9 peptides. The presence of P2X at the C-terminus enhances binding of Au NPs to cells displaying GBP1 and GBP2 peptides. This enhancement appears to be additive and suggests cooperative interactions from gold binding peptides at the N-terminus and P2X at the C-terminus. This is quantified in Figure 5 and Table S1, where statistics have been calculated and graphed for nanoparticle cluster coverage area per total cell area for each of the binding sequences as an 18 | P a g e


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 36

indicator of binding efficiency. In Figure 5a it is seen that the presence of P2X has the effect of mitigating differences in binding efficiency between the peptide sequences. When P2X is removed as in Figure 5b, the sequences break into tiers where M6G9 ~ H6G9 > GBP1 ~ GBP2. The difference in binding affinity to Au NPs could be affected by sequence dependent interaction specifics as well as peptide length and peptide flexibility. Further uncoupling of the relative binding affinity of these sequences and the interplay between the main binding peptide sequence and P2X, or other peptides inserted in place of P2X at the C-terminus, is left to future work; however, it is clear that for all the gold binding sequences tested, it is possible to induce peptidedirected assembly.

a

b

d

e

c

Figure 4. TEM images showing that binding of bacterial cells, without C-terminal P2X peptide, to 20 nm Au nanoparticles. (a) NC, (b) M6G9, (c) H6G9, (d) GBP1, (e)GBP2. Statistical data shown in Figure 5b and Table S1.

19 | P a g e


Page 21 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

Figure 5. Plots of NP coverage area/total cell area for (a) cells with P2X peptide binding to 20 nm Au NPs (where the notation “(U-M6G9-P2X)” represents un-induced cells containing M6G9-P2X plasmid but not expressing the peptide), (b) cells without P2X binding to 20 nm Au NPs, (c) cells with or without P2X as indicated, binding to 10 nm Au NPs. The value indicated by each bar corresponds to the average coverage derived from multiple cells. The error bars represent +/- 1 standard deviation.

The surface binding of bacterial cells with displayed peptides to smaller Au NPs (e.g. 10 nm) was also evaluated. The TEM images in Figure 6 display the attachment of 10 nm Au NPs to cells expressing NC, M6G9 and GBP1 peptides and include both variants: with and without codisplayed P2X. Similarly to 20 nm Au NPs, NC cells bind only a few 10 nm nanoparticles, some of which are likely from the deposition of unbound nanoparticles in the cell mixture during TEM sample preparation. In contrast to NC cells, both M6G9 and GBP1 carry high populations of nanoparticles on the cells. The presence of P2X on the M6G9-P2X or GBP1-P2X cells does not increase the binding level of 10 nm Au NPs on the cells, indicating sufficient binding affinity of M6G9 or GBP1 peptide to the 10 nm Au NPs. The P2X peptide alone can effectively bring a similar number of 10 nm Au NPs to the cell surface, as demonstrated in the representative image of NC-P2X presented at the cell surface (Figure 6b). The quantity of bound 10 nm Au NPs (Figure 6) is notably higher than that of 20 nm Au NPs (Figures 2-4) when comparing cells displaying the same binding peptide for each condition. This is also evident in Figure 5 where we note that the

20 | P a g e


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 22 of 36

coverage ratio for the 10 nm Au NPs is significantly higher than for the 20 nm Au NPs (note increased Y-axis scale in Figure 5c). As previously discussed, the displacement of the surface citrate anions leads to attachment of Au NPs at the cell surface due to reduction of electrostatic repulsion (Figure 2d). It should be emphasized that citrate anions on Au NPs are only partially exchanged with peptides, considering the short length of binding peptides in the range of several nanometers and the larger surface area of the nanoparticles. For a single nanoparticle, a 10 nm Au NP has only one-fourth the surface area of a 20 nm Au NP. Repulsive forces would be smaller in general with smaller particles. The relatively higher proportion of citrate displacement and resulting charge reduction upon peptide binding to 10 nm Au NPs versus 20 nm Au NPs, and ensuing reduction in repulsive force between the cell and the 10 nm Au NPs, would theoretically result in comparatively higher numbers of 10 nm Au NPs attached to the cells, even for slightly weaker binding peptides. In addition, from analysis of the Au NP phase map, we note that at the temperatures probed in this study 10 nm and 20 nm sized particles are expected to display morphological differences which may influence binding.48 It is also conceivable that there are sizedependences in the binding kinetics which are left to future work. So et al. investigated the relative binding of GBP1 vs triply-repeated sequence 3rGBP1 to the gold surface and observed a two-fold increase in binding affinity for 3rGBP1.49 Increasing the length of the peptides by using repeated sequences, or utilizing the C-terminus for expression of a second copy of each peptide, could possibly enhance the binding affinity of the surface-displayed peptides further as well as the loading level of relatively large Au NPs on the cells.

21 | P a g e


Page 23 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

a

b

c

d

e

f

Figure 6. TEM images showing binding of 10 nm Au NPs to the bacterial cells (a) NC, (b) NCP2X, (c) M6G9, (d) M6G9-P2X, (e) GBP1, (f) GBP1-P2X. Statistical data shown in Figure 5c and Table S1. When cell division is suppressed with the addition of a small amount (10 µg/mL) of the βlactam antibiotic aztreonam, bacterial cells can continue to elongate and form filaments as a result of inhibiting FtsI. This process has been shown to have little, if any, effect on gene expression in E. coli.50 Using the same procedures described above for unfilamented cells, 10 nm Au NPs were loaded onto the M6G9-P2X expressing cells after filamentation. Figure 7 demonstrates that these filaments carry high populations of nanoparticles, similar in density to Au NPs bound to individual E. coli cells (Figure 6d). The nanoparticles distribute uniformly across the entire filament, indicating that the binding affinity is persistent even after filamentation.

22 | P a g e


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 36

b

a

Figure 7. TEM Images demonstrating binding of 10 nm Au NPs to the M6G9-P2X filaments (ab). (a) Lower magnification. (b) Higher magnification.

Viability of Bacteria with Au NPs The deposition of nanoparticles onto bacterial cells may have potentially adverse effects, which may severely reduce cell viability.2 To investigate the viability of E. coli cells after loading gold nanoparticles, the dye pair SYTO-9–propidium iodide was used to stain the cells as a live/dead assay, followed by visualization of cells stained with each dye using fluorescence microscopy. Propidium iodide enters the cells with compromised membranes and intercalates into nucleic acids, staining non-viable cells selectively with red fluorescence, whereas SYTO-9 stains living bacteria with intact cell membranes with green fluorescence. Figure 8a-d shows examples of fluorescence microscopy images of stained bacterial cells (with displayed binding peptide and P2X) with 20 nm and 10 nm Au NPs. In the images for all investigated samples bound to Au NPs (Figures 8 and S3a-c), most of the cells fluoresce green (live), whereas only a few cells fluoresce red (dead/compromised). This demonstrates that the process of loading these Au NPs on the cell surface has only minor effects on cell viability. Quantification of cell viability was determined by flow cytometry for bacterial cells codisplaying each peptide and P2X with 20 nm Au NPs or 10 nm Au NPs, using propidium iodide

23 | P a g e


Page 25 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

staining to differentiate dead cells from live cells (Figures 8e, S4). Additionally, viability of cells with 20 nm Au NPs was performed by counting live (green) versus dead (red) cells from fluorescent images such as those represented in Figures 8 and S3 using ImageJ, which was consistent with the flow cytometry result. Figure 8e shows the viability of several cell strains with different surface peptides and different sized Au NPs. The level of viability was normalized to the viability of control cells without Au NPs. All the cell strains tested have over 95% normalized viability after loading with either size of Au NPs. Although M6G9-P2X carried many more 20 nm Au NPs as compared to the negative control, the NC-P2X and M6G9-P2X hybrid cells have similar viability, with viability for each at about 95% or higher. Similarly, M6G9-P2X and GBP1-P2X carried a higher population of 10 nm Au NPs than they did 20 nm Au NPs, and the normalized viabilities of the cells were not significantly deteriorated. Therefore, the number of citratestabilized Au NPs bound to the cells does not seem to significantly reduce cell viability. This was also true after 24 hr incubation of M6G9-P2X cells highly decorated with 10 nm Au NPs, as assessed and quantified in a similar FACS assay with propidium iodide incubation, where normalized % viability was above 90% (Figure S5).

24 | P a g e


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a

b

c

d

Page 26 of 36

e

Figure 8. Viability of E. coli displaying peptides (a-b) M6G9-P2X, (c) GBP1-P2X and (d) GBP2P2X after 1 hour incubation with and binding to 20 nm Au NPs (a, c-d) or 10 nm Au NPs (b). Cells with green fluorescence are representative of live cells, while red fluorescing cells are representative of dead or compromised cells. (e) Quantification of hybrid cell viability for various strains (displaying binding peptide and P2X) after incubation with propidium iodide and measurement of red fluorescence using FACS. Values shown were calculated by normalizing to the viability of the same cell strain without incubation with the 10 nm (blue bars) or 20 nm (green bars) Au NPs. 10,000 cells were analyzed in each case.

In addition to the cell viability tests utilizing live/dead stains, the bioactivity of functionalized cells was assessed by monitoring the cell growth rate after binding to Au NPs. M6G9-P2X displaying cells with and without 20 nm Au nanoparticles were transferred to LBcm25 medium and

25 | P a g e


Page 27 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

grown at 37 °C for 3.5 hours. The optical density (OD600) of these cells was measured at several time intervals after re-inoculation. The optical interference of Au NPs on bacteria at OD600 was minimal, in the range of 0.0 to 0.01, due to dilution during the assembly process and further dilution in medium during regrowth, and therefore OD correction to the growth curve of bacteria/Au NP hybrid cells was not necessary. Figure 9a shows that M6G9-P2X displaying cells with and without Au NPs have similar growth rates. This indicates that assembling nanoparticles with peptides displayed on the cell surface using eCPX does not affect the ability of the cells to divide or replicate. The ability of M6G9-P2X cells after binding to 10 nm Au NPs to grow and divide on agar plates and liquid culture medium was also assessed and compared to overnight growth level for bare M6G9-P2X cells (Figure S6). Even when bound to 10 nm Au NPs, these cells grew to form a lawn of bacteria on agar plates and grew to a similar OD600 as bare cells in liquid culture (Figure S6). The hybrid cells were taken out at different intervals of incubation for TEM imaging. Figure 9b shows an example of M6G9 cells with 10 nm Au NPs after one hour of growth. The density of Au NPs for each individual cell decreases with each cell division, and the bound NPs distribute evenly across the daughter cells. This phenomenon is consistent with reported artificial magnetic bacteria, where the number of magnetic nanoparticles was equally shared among the daughter bacteria during bacteria division.51 These results further demonstrate that bound Au NPs do not significantly impede cell division.

26 | P a g e


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

a

Page 28 of 36

b

Figure 9. (a) Growth curves of M6G9-P2X cells alone (blue line) and M6G9-P2X cells with 20 nm Au NPs (orange line) in LBcm25 medium demonstrate that the loading of Au NPs does not affect cell replication. (b) Induced M6G9 cells with 10 nm Au NPs after one hour of regrowth in fresh LBcm25 medium and no further induction of new peptide expression.

Metal nanoparticles have been widely investigated as one category of antimicrobial agents to protect from bacterial infection.52 The unique feature of this study is that the viability of the E. coli cells was highly conserved after assembly with Au NPs, even with a high loading of Au NPs. Several factors, such as inertness of Au (does not release metal cations), size of Au NPs, immobilization of Au NPs on cell surfaces, bio-friendly ligands and lack of toxic chemicals in the assembly process, could contribute to the high viability of the bacteria. A recent study53 demonstrated the size effect of Au NPs and Au nanoclusters on the antimicrobial properties of Gram-negative and Gram-positive bacteria. While the Au nanoclusters with a size less than 2 nm killed approximately ∼96% of the E. coli population after 2 h treatment, the free and carboxylic

acid-capped Au NPs with a size of ∼6.0 ± 3.0 nm could only kill ∼2% of the bacteria.53 In this study, Au NPs have sizes of 10 nm and above and are considered “inert” to bacteria. Moreover,

they were fixed to the cell surface due to the metal affinity for the surface displayed peptides, therefore greatly reducing the possibility of permeation of the free nanoparticles through the cell membrane, which would result in reduced cell viability. Most importantly, the nanoparticles carry the anionic ligand citrate, which is considered to be more bio-friendly than positive ligands and 27 | P a g e


Page 29 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

significantly contributes to the high viability of the hybrid cells. In some previous studies, electrostatic interactions were utilized to deposit cationic nanoparticles onto the cell’s negatively charged surface.6 However, the deposition of cationic nanoparticles potentially deteriorates the cells12, 54. Feng et al. investigated the impacts of gold nanoparticle charge and ligand type on surface binding and toxicity to Gram-negative and Gram-positive bacteria.54 The data demonstrated that cationic, especially polyelectrolyte-wrapped Au NPs, were more toxic to both the Gram-negative and Gram-positive bacteria than are negatively charged NPs. Although success in maintenance of cell viability has been achieved for the gram-positive species Bacillus cereus after deposition of poly(L-lysine)-coated 30-nm Au NPs,10 this procedure may not maintain viability in gram-negative bacteria, and the approach to Au NP deposition described herein differs in that the cells, rather than the nanoparticle, display the binding peptide, and do so in an inducible manner. Alternatively, the deposition of nanomaterials onto cells can be implemented by in situ synthesis of nanoparticles on bare cells. Biosynthesis of gold nanoparticles usually involves using gold precursors such as HAuCl4 and NaAuCl4 in the presence of cells with or without a reducing agent. However, these ionic precursors bring adverse effects to the viability of E. coli cells. We have evaluated viability of M6G9-P2X displaying E. coli cells incubated with 1 mM HAuCl4 in PBS for 1 hr. Virtually all the cells incubated with 1 mM gold salt fluoresce red after propidium iodide staining (Figure S3d), and no colonies were seen on an agar spread plate (Figure S6c). Although a lack of viability of the E. coli encased in a gold nanoshell in the work of Kuo et al., as a result of treatment with gold salt, would not negatively impact their application,12 our results suggest that the treatment of E. coli with 1 mM HAuCl4 in PBS (5x lower concentration than was described in Kuo et al) would have killed the bacterial cells. Unlike biosynthesis, our process 28 | P a g e


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 30 of 36

utilizing citrate-stabilized Au NPs is simple and does not involve any toxic chemicals. The preformed, bio-friendly nanoparticles were able to attach to the bacteria, utilizing surface displayed peptides as affinity binders for capturing the negatively charged nanoparticles.

CONCLUSIONS In summary, we have developed a straightforward strategy for attachment of pre-formed nanoparticles to bacteria which is mediated by cell surface-displayed peptides. Particularly, metalpeptide affinity driven assembly has been explored to associate metallic nanoparticles onto the surface of E. coli. Gold-binding peptides and peptides with designed sequences containing goldbinding amino acids have been integrated with the eCPX scaffold and expressed as cell surface affinity binders for nanoparticle capture. The experiments herein demonstrate successful loading of the citrate-stabilized Au NPs with the extent of capture correlated to the binding affinity of each specific peptide for Au NPs. We have also demonstrated that 10 nm Au NPs are able to bind in higher number than are 20 nm Au NPs for the peptides assessed here. More importantly, this bioconjugation strategy and the number of attached Au NPs does not significantly alter cell viability or the ability to divide. Our strategy differs from approaches that manipulate the nanoparticles themselves rather than engineering the bacteria given that induction of peptide display is required for Au NP binding in this method and therefore binding could be “turned on” as desired. Although the current study presents the assembly of bacteria with gold nanoparticles as an example, the approach can be readily generalized for conjugation of bacterial surfaces with diverse metal-rich nanoparticles, such as magnetic nanoparticles, as long as the peptide expressed meets the affinity and specificity requirements for the specific application. Furthermore, the ability to 29 | P a g e


Page 31 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

express a desired peptide sequence as linker molecules allows bacteria to be living cargo carriers and ferry many other nanomaterials and functional molecules, utilizing physical interactions or chemical linkage of peptides to these materials. The use of peptide display therefore makes this method extendible to countless applications, such as living magnetic bacteria and cargo-carrying bacteria for targeted delivery and treatment. The accessibility of both the N-terminus and Cterminus for peptide expression creates the possibility to enhance binding by expressing the same peptide at both ends of the eCPX scaffold or to create a dual binding system by expressing different peptides at the N-and C-termini, such as peptides able to recognize two distinct materials. Because the peptides are displayed on the surface of a living, self-replicating organism using induciblycontrolled peptide expression, these cells have the potential to be used as controllable living hybrids in a variety of applications.

ASSOCIATED CONTENT Supporting Information Cloning of E. coli expression plasmids, assessment of eCPX scaffold expression, procedures of particle analysis on bacterial cells, quantification of nanoparticles on hybrid cells, FACS plots for viability quantification, microscopy images of live/dead cells, and assessment of cell viability and regrowth after incubation with 10 nm Au NPs or 1 mM HAuCl4, are provided in the supporting information.

30 | P a g e


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 36

ACKNOWLEDGMENTS The authors would like to thank P. Daugherty and his laboratory for the eCPX bacterial display library that was modified and used here for peptide display, and for providing the information required to produce YPet Mona. J. Terrell, J. Jahnke and B. Adams at Army Research Laboratory (ARL) are acknowledged for useful discussions and prior related studies. S. Walck at ARL is acknowledged for help with TEM examinations. This work was performed in connection with contract W911QX-15-D-0023 with the U.S. Army Research Laboratory.

REFERENCES

(1) Chen, A. Y.; Zhong, C.; Lu, T. K., Engineering living functional materials. ACS Synth. Biol. 2015, 4, 8-11. (2) Fakhrullin, R. F.; Zamaleeva, A. I.; Minullina, R. T.; Konnova, S. A.; Paunov, V. N., Cyborg cells: functionalisation of living cells with polymers and nanomaterials. Chem. Soc. Rev. 2012, 41 (11), 4189-4206. (3) Kharlampieva, E.; Kozlovskaya, V., Cytocompatibility and Toxicity of Functional Coatings Engineered at Cell Surfaces. In Cell Surface Engineering; Fabrication of Functional Nanoshells, Fakhrullin, R. F.; Choi, I. S.; Lvov, Y., Eds. Royal Society of Chemistry: Cambridge, UK, 2014. (4) Park, J. H.; Yang, S. H.; Lee, J.; Ko, E. H.; Hong, D.; Choi, I. S., Nanocoating of Single Cells: From Maintenance of Cell Viability to Manipulation of Cellular Activities. Adv. Mater. 2014, 26, 20012010. (5) Badwaik, V. D.; Bartonojo, J. J.; Evans, J. W.; Sahi, S. V.; Willis, C. B.; Dakshinamurthy, R., Single-step biofriendly synthesis of surface modifiable, near-spherical gold nanoparticles for applications in biological detection and catalysis. Langmuir 2011, 27 (9), 5549-5554. (6) Berry, V.; Gole, A.; Kundu, S.; Murphy, C. J.; Saraf, R. F., Deposition of CTAB-terminated nanorods on bacteria to form highly conducting hybrid systems. J. Am. Chem. Soc. 2005, 127 (50), 17600-17601. (7) Huang, J.; Lin, L.; Sun, D.; Chen, H.; Yang, D.; Li, Q., Bio-inspired synthesis of metal nanomaterials and applications. Chem. Soc. Rev. 2015, 44 (17), 6330-6374. (8) Jahnke, J. P.; Cornejo, J. A.; Sumner, J. J.; Schuler, A. J.; Atanassov, P.; Ista, L. K., Conjugated gold nanoparticles as a tool for probing the bacterial cell envelope: The case of Shewanella oneidensis MR-1. Biointerphases 2016, 11 (1), 011003. (9) Berry, V.; Rangaswamy, S.; Saraf, R., Highly selective, electrically conductive monolayer of nanoparticles on live bacteria. Nano Lett. 2004, 4 (5), 939-942. (10) Berry, V.; Saraf, R. F., Self‐assembly of nanoparticles on live bacterium: an avenue to fabricate electronic devices. Angew. Chem. Int. Ed. 2005, 44 (41), 6668-6673. (11) Voet, D.; Voet, J. G., Biochemistry - 2nd Edition. John Wiley and Sons, Inc.: New York, 1995.

31 | P a g e


Page 33 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(12) Goodman, C. M.; McCusker, C. D.; Yilmaz, T.; Rotello, V. M., Toxicity of gold nanoparticles functionalized with cationic and anionic side chains. Bioconjugate Chem. 2004, 15 (4), 897-900. (13) Park, T. J.; Lee, S. Y.; Heo, N. S.; Seo, T. S., In vivo synthesis of diverse metal nanoparticles by recombinant Escherichia coli. Angew. Chem. Int. Ed. 2010, 49 (39), 7019-7024. (14) Kuo, W.-s.; Wu, C.-M.; Yang, Z.-S.; Chen, S.-Y.; Chen, C.-Y.; Huang, C.-C.; Li, W.-M.; Sun, C.-K.; Yeh, C.-S., Biocompatible bacteria@Au composites for Application in the photothermal destruction of cancer cells. Chem. Commun. 2008, 4430-4432. (15) Kahraman, M.; Zamaleeva, A. I.; Fahkrullin, R. F.; Culha, M., Layer-by-layer coating of bacteria with noble metal nanoparticles for surface-enhance Raman scattering. Analytical and Bioanalytical Chemistry 2009, 395, 2559-2567. (16) Vedantum, P.; Tzeng, T.-R. J.; Brown, A. K.; Podila, R.; Rao, A.; Staley, K., Binding of Escherichia coli to Functionalized Gold Nanoparticles. Plasmonics 2012, 7, 301-308. (17) Chen, A. Y.; Deng, Z.; Billings, A. N.; Seker, U. O.; Lu, M. Y.; Citorik, R. J.; Zakeri, B.; Lu, T. K., Synthesis and patterning of tunable multiscale materials with engineered cells. Nat. Mater. 2014, 13 (5), 515-523. (18) Chaudhary, P. M.; Sangabathuni, S.; Murthy, R. V.; Paul, A.; Thulasriam, H. V.; Kikkeri, R., Assessing the effect of different shapes of glyco-gold nanoparticles on bacterial adhesion and infections. Chem. Commun. 2015, 51, 15669-15672. (19) Daugherty, P. S., Protein engineering with bacterial display. Curr. Opin. Struc. Biol. 2007, 17 (4), 474-480. (20) Rice, J. J.; Schohn, A.; Bessette, P. H.; Boulware, K. T.; Daugherty, P. S., Bacterial display using circularly permuted outer membrane protein OmpX yields high affinity peptide ligands. Protein Sci. 2006, 15 (4), 825-836. (21) Rice, J. J.; Daugherty, P. S., Directed evolution of a biterminal bacterial display scaffold enhances the display of diverse peptides. Protein Eng. Des. Sel. 2008, 21 (7), 435-442. (22) Getz, J. A.; Rice, J. J.; Daugherty, P. S., Protease-resistant peptide ligands from a knottin scaffold library. ACS Chem. Biol. 2011, 6 (8), 837-844. (23) Sarkes, D. A.; Dorsey, B. L.; Finch, A. S.; Stratis-Cullum, D. N., Method for Discovery of Peptide Reagents Using a Commercial Magnetic Separation Platform and Bacterial Cell Surface Display Technology. J. Anal. Bioanal. Tech. 2015, 6 (4), 1. (24) Sarkes, D. A.; Hurley, M. M.; Stratis-Cullum, D. N., Unraveling the Roots of Selectivity of Peptide Affinity Reagents for Structurally Similar Ribosomal Inactivating Protein Derivatives. Molecules 2016, 21 (11), 1504. (25) Kogot, J. M.; Pennington, J. M.; Sarkes, D. A.; Kingery, D. A.; Pellegrino, P. M.; Stratis‐Cullum, D. N., Screening and characterization of anti‐SEB peptides using a bacterial display library and microfluidic magnetic sorting. J. Mol. Recognit. 2014, 27 (12), 739-745. (26) Sarkes, D. A.; Jahnke, J. P.; Stratis-Cullum, D. N., Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates. J. Vis. Exp. 2017, e56061. (27) Adams, B. L.; Finch, A. S.; Hurley, M. M.; Sarkes, D. A.; Stratis‐Cullum, D. N., Genetically engineered peptides for inorganics: study of an unconstrained bacterial display technology and bulk aluminum alloy. Adv. Mater. 2013, 25 (33), 4585-4591. (28) Adams, B. L.; Hurley, M. M.; Jahnke, J. P.; Stratis-Cullum, D. N., Functional and Selective Bacterial Interfaces Using Cross-Scaffold Gold Binding Peptides. JOM 2015, 67 (11), 2483-2493. (29) Sankaran, S.; Kiren, M. C.; Jonkheijm, P., Incorporating bacteria as a living component in supramolecular self-assembled monolayers through dynamic nanoscale interactions. ACS nano 2015, 9 (4), 3579-3586. (30) Jabaiah, A. M.; Getz, J. A.; Witkowski, W. A.; Hardy, J. A.; Daugherty, P. S., Identifi cation of protease exosite-interacting peptides that enhance substrate cleavage kinetics. Biol. Chem. 2012, 9, 933941. (31) Brown, S., Metal-recognition by repeating polypeptides. Nat. Biotechnol. 1997, 15 (3), 269-272. 32 | P a g e


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 36

(32) Sarikaya, M.; Tamerler, C.; Jen, A. K.-Y.; Schulten, K.; Baneyx, F., Molecular biomimetics: nanotechnology through biology. Nat. Mater. 2003, 2 (9), 577-585. (33) Seker, U. O. S.; Demir, H. V., Material binding peptides for nanotechnology. Molecules 2011, 16 (2), 1426-1451. (34) Tan, Y. N.; Lee, J. Y.; Wang, D. I., Uncovering the design rules for peptide synthesis of metal nanoparticles. J. Am. Chem. Soc. 2010, 132 (16), 5677-5686. (35) Peelle, B. R.; Krauland, E. M.; Wittrup, K. D.; Belcher, A. M., Design criteria for engineering inorganic material-specific peptides. Langmuir 2005, 21 (15), 6929-6933. (36) Okada, Y.; Takano, T. Y.; Kobayashi, N.; Hayashi, A.; Yonekura, M.; Nishiyama, Y.; Abe, T.; Yoshida, T.; Yamamoto, T. A.; Seino, S., New protein purification system using gold-magnetic beads and a novel peptide tag,“the methionine tag”. Bioconjugate Chem. 2011, 22 (5), 887-893. (37) Aldeek, F.; Safi, M.; Zhan, N.; Palui, G.; Mattoussi, H., Understanding the self-assembly of proteins onto gold nanoparticles and quantum dots driven by metal-histidine coordination. ACS nano 2013, 7 (11), 10197-10210. (38) Albanese, A.; Chan, W. C. W., Effect of Gold Nanoparticle Aggregation on Cell Uptake and Toxicity. ACS Nano 2011, 5 (7), 5478-5489. (39) Schindelin, J.; Arganda-Carreras, I.; Frise, E.; Kaynig, V.; Longair, M.; Pietzsch, T.; Preibisch, S.; Rueden, C.; Saalfeld, S.; Schmid, B., Fiji: an open-source platform for biological-image analysis. Nat. Methods 2012, 9 (7), 676-682. (40) Schindelin, J.; Rueden, C. T.; Hiner, M. C.; Eliceiri, K. W., The ImageJ ecosystem: An open platform for biomedical image analysis. Mol. Reprod. Dev. 2015, 82 (7-8), 518-529. (41) van der Walt, S.; Colbert, S. C.; Varoquaux, G., The NumPy array: a structure for efficient numerical computation. Comput. Sci. Eng. 2011, 13 (2), 22-30. (42) Jones, E.; Oliphant, T.; Peterson, P., {SciPy}: open source scientific tools for {Python}. http://www.scipy.org/ 2017, Version 0.19. (43) Verde, A. V.; Acres, J. M.; Maranas, J. K., Investigating the specificity of peptide adsorption on gold using molecular dynamics simulations. Biomacromolecules 2009, 10 (8), 2118-2128. (44) Corni, S.; Hnilova, M.; Tamerler, C.; Sarikaya, M., Conformational Behavior of GeneticallyEngineered Dodecapeptides as a Determinant of Binding Affinity for Gold. The Journal of Physical Chemistry C 2013, 117 (33), 16990-17003. (45) Seker, U. O. S.; Wilson, B.; Kulp, J. L.; Evans, J. S.; Tamerler, C.; Sarikaya, M., Thermodynamics of Engineered Gold Binding Peptides: Establishing the Structure–Activity Relationships. Biomacromolecules 2014, 15 (7), 2369-2377. (46) Rice, J. J., Development and optimization of bacterial display methodologies for peptide library screening. University of California, Santa Barbara: 2007. (47) Singer, S. J.; Nicolson, G. L., The fluid mosaic model of the structure of cell membranes. Science 1972, 175 (4023), 720-731. (48) Barnard, A. S.; Young, N. P.; Kirkland, A. I.; Van Huis, M. A.; Xu, H., Nanogold: a quantitative phase map. ACS nano 2009, 3 (6), 1431-1436. (49) So, C. R.; Tamerler, C.; Sarikaya, M., Adsorption, Diffusion, and Self‐Assembly of an Engineered Gold‐Binding Peptide on Au (111) Investigated by Atomic Force Microscopy. Angew. Chem. Int. Ed. 2009, 48 (28), 5174-5177. (50) Arends, S. R.; Weiss, D. S., Inhibiting cell division in Escherichia coli has little if any effect on gene expression. Journal of bacteriology 2004, 186 (3), 880-884. (51) Martín, M.; Garcés, V.; Domínguez-Vera, J. M.; Gálvez, N., Magnetism in living magneticallyinduced bacteria. RSC Advances 2016, 6 (97), 95220-95226. (52) Slavin, Y. N.; Asnis, J.; Häfeli, U. O.; Bach, H., Metal nanoparticles: understanding the mechanisms behind antibacterial activity. J. Nanobiotechnol. 2017, 15, 65. (53) Zheng, K.; Setyawati, M. I.; Leong, D. T.; Xie, J., Antimicrobial Gold Nanoclusters. ACS nano 2017, 11 (7), 6904-6910. 33 | P a g e


Page 35 of 36 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

(54) Feng, Z. V.; Gunsolus, I. L.; Qiu, T. A.; Hurley, K. R.; Nyberg, L. H.; Frew, H.; Johnson, K. P.; Vartanian, A. M.; Jacob, L. M.; Lohse, S. E., Impacts of gold nanoparticle charge and ligand type on surface binding and toxicity to Gram-negative and Gram-positive bacteria. Chem. Sci. 2015, 6 (9), 51865196.

34 | P a g e


Langmuir 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 36

For Table of Contents Use Only

35 | P a g e


Living Bacteria-Nanoparticle Hybrids Mediated through Surface

Recommend Documents