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Article Cite This: ACS Omega 2018, 3, 16489−16499
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Screening of a Cell-Penetrating Peptide Library in Escherichia coli: Relationship between Cell Penetration Efficiency and Cytotoxicity Kazusato Oikawa, Md Monirul Islam, Yoko Horii, Takeshi Yoshizumi, and Keiji Numata* Biomacromolecules Research Team, RIKEN Center for Sustainable Resource Science, 2-1 Hirosawa, Wako-shi, Saitama 351-019, Japan
ACS Omega 2018.3:16489-16499. Downloaded from pubs.acs.org by 185.101.69.63 on 12/06/18. For personal use only.
S Supporting Information *
ABSTRACT: Cell-penetrating peptides (CPPs) have been used for the intracellular delivery of bio-active cargo, such as drugs, genes, and proteins. Similarly, CPPs have been successfully used against bacteria, viruses, and yeasts over the past few decades. To broaden the possible applications of CPPs, controlling the balance between their cell-penetrating efficiency and their cytotoxicity to target cells is essential. Here, we aim to identify a CPP for bacteria that has a high penetration efficiency and low cytotoxicity. We assayed a library of 55 CPPs for Escherichia coli (E. coli) DH5α using a combination of fluorescence spectroscopy (FS) and confocal laser scanning microscopy (CLSM). We found that several cationic CPPs, such as R9-TAT, (KH)9, and Rev (34−50), and amphipathic CPPs, such as ppTG1 and pAntpHD (Pro50), have the highest penetration efficiency in E. coli DH5α and low cytotoxicity. The analysis of the cellular penetration of E. coli DH5α by TAMRA-labeled CPPs using two independent techniques, namely, FS and CLSM, and double staining with FM4-64, which is a cell membrane-staining dye, yielded quantitative and qualitative results, confirming that TAMRA-labeled CPPs accumulate in the E. coli cytoplasm. This study provides peptide library-based information of CPPs with high bacterial cell penetration efficiency and low cytotoxicity.
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the lipid components of the membrane,5,14 which affects their bactericidal activity and cytotoxicity.15 Hydrophobic CPPs possess only a nonpolar face that is considered the major force of the interaction with the lipid surface of the cell membrane.5 In addition to their charge, the secondary structure of CPPs is a key factor that determines their penetration efficiency.16 Hydrophobic amino acids present in helical sequences cause the peptides to form helical structures, affecting their membrane penetration efficiency.17 The cationic α-helical structure of CPPs destabilizes the structure of the plasma membranes.18 A recent report showed that the stable α-helical structure of amphipathic CPPs enhances their penetration efficiency in HeLa cells.19 Additionally, some CPPs have been reported to exhibit a negligible secondary structure in aqueous solution but change their conformation to an α-helical structure during their association with sodium dodecyl sulfate (SDS), which is used to mimic the cell membrane conditions.20 To evaluate the secondary structures of CPPs in cell membranes, cell membrane-mimicking conditions, such as incubation in dilute SDS, have been used by many research groups.21
INTRODUCTION Cell-penetrating peptides (CPPs) have been historically discovered as unique cationic peptides that exhibit relatively low cytotoxicity and are basically different from cationic antimicrobial peptides that generally exhibit cytotoxicity with perturbation on cell membranes.1,2 However, in this study, to avoid confusion related to the historical and scientific classification of CPP and cationic antimicrobial peptides, we call both the peptides penetrating the plasma membrane as CPP. CPPs are short peptides consisting of less than 40 amino acids. CPPs are classified into the following three categories: cationic, amphipathic, and hydrophobic.3−5 In general, cationic CPPs can enter cells by interacting with the negatively charged bacterial membrane.6−9 The presence of cationic amino acid residues, such as lysine (Lys) and arginine (Arg), greatly affects the cell membrane penetration efficiency of CPPs.10 However, cationic CPPs with positive charges display increased cytotoxicity because of the membrane destruction.11 These CPPs bind electrostatically to the bacterial membrane, leading to non-enzymatic disruption through a pore-formation mechanism12 that occurs because the bacterial membranes are more negatively charged than the mammalian cell membranes because of the higher content of anionic phospholipids, lipoteichoic acid, and lipopolysaccharide.13 Amphipathic CPPs possess a hydrophilic face that binds phospholipid head groups and a hydrophobic face that binds © 2018 American Chemical Society
Received: September 11, 2018 Accepted: November 21, 2018 Published: December 4, 2018 16489
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Figure 1. Uptake efficiency of TAMRA-labeled CPPs by E. coli DH5α analyzed by FS. Suspensions of E. coli DH5α at a density of 106 cells/μL were stained with CPPs at 0.1 μg/μL for 30 min. The data represent the mean ± SD (n = 5).
Escherichia coli (E. coli) DH5α as a model Gram-negative bacterium for the CPP library screening. The CPP library was previously constructed for a study investigating CPP functions in various plant cells.33 The CPPs used in this study are described by the peptide numbers and names listed in Table S1. This study provides peptide library-based information of CPPs with high bacterial cell penetration efficiency and low cytotoxicity.
The precise mechanisms by which CPPs are taken up by cells are still not completely understood. In eukaryotic cells, several direct transduction or endocytosis pathways have been recognized.22 Surface-bound CPPs penetrate the lipid bilayer, and the electrostatic interaction between the CPPs and the membrane seems to control the incorporation mechanism through pore formation.12,23 The interaction between the plasma membrane and CPPs highly depends on the composition and characteristics of the phospholipid membrane.24 Compared with mammalian cells, most bacteria contain more anionic phospholipids on the inner and outer surfaces of the membrane, which further contributes to the penetration efficiency of CPPs.25 The endocytosis of CPPs has been shown in eukaryotic cells, especially at lower CPP concentrations (below 2.0 μM, ca. 4.7 μg/mL), whereas the translocation has been detected at higher CPP concentrations (over 5.0 μM, ca. 16 μg/mL).26 However, most prokaryotic cells, such as bacteria, typically have a simple membrane system; for example, endocytosis is lacking, and no membrane vesicles are present in the cytoplasm.27 Therefore, bacterial cells may be useful for revealing the mechanism by which CPPs enter the cytosol. For example, certain CPPs, such as TP10, penetratin, and pVEC, interact with bacterial cells and damage their plasma membranes to increase the membrane permeability.28,29 In contrast, some CPPs enter the bacterial membrane without causing damage and hinder several important pathways related to DNA replication and protein synthesis.30 For instance, buforin II can enter cells and interact with DNA and RNA without causing significant damage to the cell membrane.31 A very recent report showed that the main cause of cell death due to CPP is the strong interactions between the peptides and genomic DNA rather than damage to the plasma membrane.32 Thus, the cell-penetrating efficiency and cytotoxicity of CPPs are determined by their secondary structures and amino acid sequences. Here, we attempted to clarify the correlation between the cell penetration efficiency and cytotoxicity of CPPs using a CPP library comprising 55 types of CPPs (Table S1) and
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RESULTS Fluorescence Spectroscopy-Based Cellular Uptake Efficiency. Several studies have reported that CPPs that contain lysine (Lys) and arginine (Arg) residues show more efficient uptake into eukaryotic cells.10,34 The penetration of eukaryotic mammalian and plant cells by some CPPs has been previously reported.35,36 Here, we conducted several experiments to confirm the efficiency of CPP uptake by E. coli DH5α. In the CPP library, there are CPPs with repetitive sequences. For instance, (2) 2BP100, (9) K18, and (34) 2× ppTG1 are a dimer of (1) BP100, (8) K9, and (32) ppTG1, respectively. At the same molar concentrations of those CPPs, dimers usually show higher efficiency and cytotoxicity because of the twice longer sequence. To avoid such an effect of molecular weight, we used weight concentration in this study rather than molar concentration, which is widely used for the CPP study. We calculated the CPP uptake efficiency and compared the results obtained using fluorescence spectroscopy (FS). First, TAMRA-labeled CPPs were incubated with E. coli DH5α, and their penetration efficiency was quantitatively determined. Figure S1 briefly illustrates the protocol used to measure the FS-based uptake efficiency. The FS-based uptake efficiency was determined using calibration curves (Figure S2). Figure 1 shows the ranking of the FS-based CPP uptake efficiency in E. coli DH5α. The results show that a series of Lys- and Arg-rich cationic and amphipathic CPPs exhibited the highest uptake efficiency in E. coli DH5α and that the hydrophobic CPPs were generally taken up at a lower efficiency. Of the Lys-rich cationic CPPs, (7) (KH)9 displayed 16490
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Figure 2. Representative image used in the analysis of the uptake efficiency and visualization efficiency of TAMRA-labeled CPPs by E. coli DH5α by CLSM. (A) Blank measurement. (B) E. coli DH5α at a density of 106 cells/μL were stained with CPPs at 0.1 μg/μL for 30 min. Scale bars: merged image, 5 μm; enlarged image, 2 μm. See the text for a description of the experimental protocol.
Figure 3. Quantitative analysis of the uptake efficiency of TAMRA-labeled CPPs by E. coli DH5α by CLSM. E. coli DH5α at a density of 106 cells/ μL were stained with CPPs at 0.1 μg/μL for 30 min. The data represent the mean ± SD (n = 5).
Amphipathic CPPs may better penetrate the E. coli cell membrane because they bind phospholipids and lipopolysaccharides because of their structural properties, namely, hydrophobicity and helicity.37 One amphipathic CPP, that is, (32) ppTG1, showed approximately 16.0% penetration efficiency. Another amphipathic CPP containing both Lys and Arg residues, that is, (35) pAntpHD (Pro50), showed a penetration efficiency of 11.0% in E. coli. In contrast, an amphipathic histidine (His)-rich CPP, that is, (28) buforin II (5−21), could penetrate the cell membrane as previously reported.38 In this study, this His-rich CPP showed a penetration efficiency of 6.0%, which is relatively lower than that of Lys- and Arg-rich CPPs. The other amphipathic CPP, that is, (46) CytC 71−101, demonstrated a lower penetration efficiency0.4%even though it contains 6 Lys residues. The hydrophobic CPP (33) transportan (TP) displayed approximately 6.6% penetration efficiency. (31) LAH4, which is a well-known hydrophobic CPP containing 26 amino acid residues, including four His residues,39 showed a penetration efficiency of 4.6%, which is lower than that of the Lys- and Argrich CPPs. Furthermore, several CPPs, including (41) Inv3, (43) Inv3.5, and (42) Inv5, showed negligible penetration
the highest efficiency (approximately 22%) in the FS experiment. The cationic charge of (7) (KH)9 may confer superior ability to bind electrostatically to the negatively charged phospholipids that constitute the bacterial membrane; consequently, this CPP can form pores in the bacterial membrane and move inside the bacterium.12 The other Lysrich cationic CPPs, that is, (2) 2Bp100, (11) DPV3, and (9) K18, exhibited 9.4, 8.8, and 7.8% penetration efficiency, respectively. Of the Arg-rich cationic CPPs, (13) R9-TAT showed the highest penetration efficiency (12.3%) in E. coli. The other Arg-rich cationic CPPs, that is, (3) Rev (34−50) and (6) R12, showed 9.4 and 5.9% penetration efficiency, respectively. Remarkably, the penetration efficiency in E. coli DH5α did not depend on the number of cationic residues in the CPP sequence. For instance, while (4) R9, (5) D-R9, (6) R12, (8) K9, and (9) K18 are highly cationically charged CPPs, they showed relatively lower penetration efficiency than (13) R9-TAT, (3) Rev, (7) (KH)9, and (11) DPV3. Thus, the cationic charge had only a minor effect, indicating that the penetration of E. coli cells by CPPs is affected by their composition, length, and structural properties. 16491
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Figure 4. Analysis of the visualization efficiency of TAMRA-labeled CPPs in E. coli DH5α. E. coli DH5α at a density of 106 cells/μL were stained with CPPs at 0.1 μg/μL for 30 min. The data represent the mean ± SD (n = 5).
composition and helical conformation. Moreover, the electrostatic repulsion between the positively charged helices of these highly charged CPPs may cause the peptides to insert into the bacteria as previously reported.12 Some cationic CPPs, such as (16) Sc18, (12) 6-Oct, and (14) Tat (49−57), showed approximately 28, 25, and 22% penetration efficiency, respectively, suggesting that the presence of an appropriate substituent in the CPP sequence is required for E. coli membrane penetration. An amphipathic CPP, that is, (32) ppTG1, displayed the second highest penetration efficiency (104%). The amphipathic CPPs contain hydrophobic faces that facilitate their binding to phospholipids and lipopolysaccharides, thereby increasing penetration efficiency.41 Moreover, (32) ppTG1 contains positively charged Lys residues on its nonpolar face, which may alter the α-helix and confer higher penetration efficiency through the cellular membrane. An amphipathic CPP, that is, (48) Glu-Oct-6, displayed a penetration efficiency (2.5%) that was lower than that of the Lys- and Arg-rich CPPs. These CPPs may kill bacteria without disrupting the membrane integrity by inhibiting important pathways, such as DNA replication and protein synthesis, within cells.42 Some CPPs, such as (18) IX, (34) 2× ppTG1 and (50) G53-4, require 10% DMSO for solubility. These CPPs may aggregate on the bacterial cell membrane and lose their ability to interact with E. coli and penetrate the membrane.42 The hydrophobic CPP (33) transportan (TP) showed penetration efficiency of approximately 43%. Another hydrophobic CPP, that is, (55) MG2d, showed an uptake efficiency of 18% as determined by the CLSM image analysis. These CPPs are cationic and hydrophobic, which may cause them to assume a helical conformation. Consequently, these CPPs exhibit increased binding to E. coli cells.10,11 However, some hydrophobic CPPs, including (19) XI, (42) Inv3, and (44) Inv3.10, showed an uptake efficiency of approximately 2%, which is quite low. Penetration Efficiency Based on Cellular Visualization. Using the CLSM images, we manually counted the penetrated cells (TAMRA-stained cells) and calculated the
efficiencies of approximately 0.5%. Although these CPPs are known protein carriers in HeLa cells,40 they did not efficiently penetrate the E. coli cell membrane. Confocal Laser Scanning Microscopy-Based Cellular Uptake Efficiency. Confocal laser scanning microscopy (CLSM) was conducted to observe CPP penetration into E. coli DH5α. Figure 2 shows a representative CLSM image of a TAMRA-labeled CPP inside a cell. To quantify the cell penetration efficiency, we visualized the CPP penetration and calculated the relative fluorescence intensity of the CLSM images using ImageJ (Figure S3). (7) (KH)9 was used as a standard to evaluate the efficiency; the fluorescence intensity obtained using (7) (KH)9 was set to 100%. Figure 3 shows the ranking of the penetration efficiency of various CPPs in E. coli DH5α based on the CLSM observations. The CLSM analyses clearly show CPP internalization within E. coli cells, whereas the FS results measure the uptake efficiency, including CPPs bound to the cell membrane and internalized CPPs. Consistent with the results obtained using FS, a series of Lys- and Arg-rich CPPs exhibited higher penetration efficiency in E. coli DH5α based on the relative fluorescence intensity measured by CLSM, whereas the hydrophobic CPPs exhibited relatively poorer penetration efficiency. An Arg-rich cationic CPP, that is, (13) R9-TAT, showed the highest efficiency (129%) by CLSM. Two other Arg-rich cationic CPPs, that is, (3) Rev (34−50) and (4) R9, exhibited 108 and 52% cell penetration efficiencies, respectively, in E. coli. A Lys-rich cationic CPP, that is, (7) (KH)9, also showed relatively high efficiency in the CLSM image analysis. Other Lys-rich cationic CPPs, such as (11) DPV3, (2) 2BP100, and (9) K18, showed penetration efficiencies of 97, 72, and 50%, respectively. These results are consistent with the relative fluorescence intensity results obtained using FS. Consistent with the FS results, the highly cationic CPPs, such as (4) R9, (5) D-R9, (6) R12, (8) K9, and (9) K18, showed relatively lower penetration efficiency. This finding indicates that the cationic nature of CPPs is not sufficient to allow the peptides to penetrate the E. coli cell membrane. In addition to the cationic property, penetration may require a diverse 16492
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Figure 5. Representative CLSM image of the localization of TAMRA-labeled CPPs in E. coli DH5α after double staining with FM4-64. (A) CLSM images of TAMRA-labeled R9-TAT localized within cells. The merged image is composed of TAMRA, FM4-64, and bright-field images. Scale bar: 2 μm. (B) Enlarged image showing the area indicated by the broken line in (A). Scale bar: 1 μm. (C) RGB profiles of the area indicated by the broken line in (B). The red line denotes a TAMRA-labeled CPP, the green line denotes FM4-64, and the blue line denotes the background. (D) 3D surface plot of E. coli treated with TAMRA-labeled R9-TAT.
dye is present around the cell membrane (green), whereas the TAMRA-labeled (13) R9-TAT is inside the cell (red). Figure 5B presents an enlarged image showing that the TAMRAlabeled CPPs accumulated within the E. coli cells and that FM4-64 surrounded the cell membrane. As shown in Figure 5C, the RGB profiles of the TAMRA-labeled cells indicate the accumulation of TAMRA-labeled CPP and the cell membrane (FM4-64). Figure 5D shows a surface plot of an E. coli cell in which TAMRA-labeled R9-TAT has accumulated; the plot indicates that the CPP has successfully penetrated the cell and is localized inside the cell membrane. As shown in Figure S4, all CPPs in the library were characterized by the same method. (28) Buforin II (5−21), which displayed moderate penetration efficiency, was found to be moderately internalized inside E. coli DH5α (Figure S4). The TAMRA-labeled CPP accumulated at low levels inside the plasma membrane. In contrast to the signals of FM4-64 on the cell membrane, the RGB profiles indicate that (28) buforin II (5−21) was partially stored within the cell. The CPPs with the lowest penetration efficiencies, such as (19) XI, showed no internalization within the E. coli cells (Figure S4). The enlarged image, 3D surface plot, and RGB profiles clearly show that (19) XI was not internalized into the E. coli cells. Cytotoxicity of CPPs. The cell viability in the presence of CPPs was evaluated using an Evans blue (EB) assay. EB cannot bind live cells but does bind dead cells44 because EB binds globular proteins or membrane proteins that are degradation products from dead cells. As shown in Figure 6A, EB bound E. coli cells killed by a 70% ethanol treatment, and as shown in Figure 6B, no EB bound live E. coli cells. In this study, all experiments were conducted at a CPP concentration of 0.1 μg/ μL; this CPP concentration was not obviously cytotoxic to E.
uptake efficiency. This method of determining uptake efficiency considers the number of positive cells rather than the amount of CPP inside the cells. Figure 4 shows the ranking of the uptake efficiency of the CPPs into E. coli DH5α as determined by CLSM. A Lys-rich cationic CPP, that is, (7) (KH)9, showed the highest cell visualization efficiency (96%). (6) R12 is Arg-rich and cationic and displays a visualization efficiency of 87%. The Lys- and Arg-rich cationic CPPs (13) R9-TAT, (2) 2Bp100, (11) DPV3 (4) R9, and (8) K9 showed visualization efficiencies of 86, 84, 83, 80, and 79%, respectively. The cationic CPPs (15) Retro-Tat (49−57), (16) Sc18, and (14) Tat (49−57) showed visualization efficiencies of 54, 53, and 52%, respectively. The amphipathic CPP (32) ppTG1 showed the second highest visualization efficiency (93%), which is consistent with the uptake efficiency of this CPP determined by other methods. The Lys- and Argrich amphipathic CPPs (35) pAntpHD (pro50), (17) KLA10, (22) PenArg, and (24) penetratin showed cell visualization efficiencies of 72, 68, and 67%, respectively, in E. coli. Remarkably, the hydrophobic CPPs (55) MG2d and (33) transportan (TP) showed visualization efficiencies of 71 and 57%, respectively. Cellular Localization. The FS and CLSM experiments showed that CPPs penetrate E. coli DH5α cells. We must determine whether the CPPs are bound to the cell membrane or internalized. The cellular localization of the CPPs was distinguished using CLSM with double staining of TAMRAlabeled CPPs and FM4-64,43 which is a membrane-staining dye. Figure 5A shows that one of the most efficiently penetrating CPPs, that is, (13) R9-TAT, showed the highest internalization in E. coli cells. The enlarged image and threedimensional (3D) surface plot clearly show that the FM4-64 16493
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Figure 6. Measurement of the cytotoxic effect of CPPs on E. coli DH5α visualized with EB by CLSM. (A) E. coli DH5α at a density of 106 cells/μL were treated with 70% ethanol for 30 min to kill the cells; then, 0.25% (v/v) EB was added for 30 min, and the cells were washed three times. (B) E. coli DH5α at a density of 106 live cells/μL were mixed with 0.25% (v/v) EB and washed three times. (C) E. coli DH5α at a density of 106 cells/ μL were stained with CPPs at 0.1 μg/μL for 30 min; then, 0.25% (v/v) EB was added for 30 min. (D) E. coli DH5α at a density of 106 cells/μL were stained with CPPs at 5 μg/μL for 30 min; then, 0.25% (v/v) EB was added for 30 min. Scale bar, 10 μm. See the text for a description of the experimental protocol.
Figure 7. Cytotoxic effect of CPPs on E. coli DH5α measured using an EB assay. E. coli DH5α at a density of 106 cells/μL were stained with CPPs at 5 μg/μL for 30 min; then, 0.25% (v/v) EB was added for 30 min. The data represent the mean ± SD (n = 50).
of 5 μg/μL, all CPPs induced cell death at rates ranging from 27 to 63%. Several CPPs, including (42) Inv5, (48) Glu-Oct-6, (51) M591, (28) buforin II (5−12), (37) Crot (27−39), and (39) Crot (27−39) derivative 2, showed relatively high cytotoxicity to E. coli (Figure 7) and low penetration efficiency (Figure 3). However, the CPPs with higher penetration efficiency, such as (13) R9-TAT, (7) (KH)9, (3) Rev (34− 50), (32) ppTG1, and (35) pAntp (44−58), showed lower cytotoxicity to E. coli.
coli cells based on EB assays at a cell density of 106 cells/μL. Therefore, we evaluated the minimum inhibitory concentration (MIC) of (7) (KH)9, which was used as a model CPP (Figure S5). The MIC was determined at 50% of the EB intensity range. As shown in Figure S6, the MIC of (7) (KH)9 against E. coli was approximately 5 μg/μL. Thus, 5 μg/μL was the minimum concentration of (7) (KH)9 required to kill E. coli DH5α. We conducted dose−response experiments using the compounds in the CPP library (Figure 7). At a concentration 16494
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Figure 8. Correlation between the FS and CLSM penetration efficiencies and cell visualization efficiencies in E. coli DH5α. (A) Correlation of the uptake efficiencies determined by FS and CLSM. The correlation plot shows a high positive correlation at both the quantitative and qualitative levels. (B) High positive correlation was observed between the CLSM uptake efficiency and cell visualization efficiency determined by the CLSM image analysis. (C) High positive correlation was observed between the FS uptake efficiency and cell visualization efficiency determined by the CLSM image analysis. All values obtained using the two independent techniques show significant differences by two-way ANOVA (*P < 0.05).
Figure 9. Correlation between the penetration efficiency of the CPPs and their cytotoxicity. (A) Negligible correlation was observed between the cytotoxicity and CLSM uptake efficiency. (B) Negligible correlation was observed between the cytotoxicity and FS uptake efficiency. All values obtained using the two independent techniques show significant differences by two-way ANOVA (*P < 0.05).
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correlation.49 A series of Lys-rich and Arg-rich cationic CPPs, including (13) R9-TAT, (7) (KH)9, and (3) Rev (34−50), showed a relatively high penetration efficiency based on the fluorescence intensity assessed by both FS and CLSM. (7) (KH)9 showed the highest efficiency in the FS experiment but a comparatively lower uptake efficiency based on the relative fluorescence intensity determined by CLSM because (7) (KH)9 preferentially attaches to the E. coli membrane rather than internalizing into the cytoplasm. Thus, we observed a relatively lower internalization by CLSM. The cell visualization efficiency, which is a measure of penetration efficiency based on the number of positive cells observed, was also positively correlated with the other experimental results (Figure 8B,C). Collectively, based on their highest penetration efficiency and their visualization efficiency as determined by CLSM, the CPPs that most efficiently penetrated E. coli DH5α were (7) (KH)9, (13) R9TAT, (32) ppTG1, (3) Rev(34−50), (11) DPV3, and (35) pAntpHD (Pro50). These peptides are highly cationic and might display helical or random coil structure during their interactions with cholesterol-containing phospholipids.16,50
DISCUSSION The efficacy of the internalization of CPPs into cells has been studied using various methods, such as FS, flow cytometry, and microscopic imaging.45 Several physicochemical parameters, including the net charge, amphipathicity, hydrophobicity, length, helicity, and solubility, are thought to affect the cell penetration efficiency and cytotoxicity of CPPs. The efficiency of the penetration of bacteria by CPPs is considered to be mainly determined by the balance among the cationicity (net positive charge), hydrophobicity, and helicity of the peptides.37,46−48 Here, we used the FS and CLSM techniques to study the internalization of CPPs and their cytotoxic effects on bacteria. The methods used in this study enabled us to quantify the membrane-bound CPPs, the CPPs internalized within E. coli DH5α and their cytotoxic effects. To obtain the quantitative results, we conducted FS experiments; the qualitative results were obtained by CLSM. The correlation between the cell-penetrating efficiency determined using the two different methods was characterized (Figure 8). The relative fluorescence intensity determined by FS and CLSM (Figure 8A) showed a high positive 16495
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differ among prokaryotic cell and eukaryotic mammalian and plant cells.13,59 Considering the current results and those previous reports, it is difficult to find any relationship between the membrane structures and CPP functions. To design an optimal CPP for target cells, we need to adopt knowledge from the long-term studies on CPP.
These characteristics play a major role in cell membrane penetration through pore-formation mechanisms.12,51−53 The correlation plot (Figure 8) indicates that Lys- and Argrich CPPs, such as (5) D-R9, (6) R12, (8) K9, and (9) K18, do not efficiently penetrate E. coli cells. Thus, the substitution of some cationic residues by hydrophobic residues is required for better penetration efficiency. A previous report has shown that the addition of a single hydrophobic residue to an Arg- or Lys-rich CPP significantly alters the membrane penetration efficiency.54 Conversely, the substitution of a highly positively charged Lys by a hydrophobic residue increases the cell penetration efficiency.17 A very recent report demonstrated that the position rather than the positive charge on the residue affects cell penetration.17 Moreover, as shown in Figure 8, CPPs with hydrophobic-rich sequences display poorer penetration efficiency in E. coli. To evaluate the cytotoxicity of individual CPPs, TAMRAlabeled CPP-treated E. coli were stained with EB. Cytotoxic CPPs may be very effective at low concentrations and kill bacteria either directly by membrane disruption or metabolic function blockade. Remarkably, as shown in Figure 9, the correlation between the penetration efficiency of the CPPs and their cytotoxicity is negligible. In this CPP library screening, we observed that some CPPs, including (42) Inv5, (48) Glu-Oct6, (51) M591, (28) buforin II (5−12), (37) Crot (27−39), and (39) Crot (27−39) derivative 2, kill E. coli cells, resulting in relatively high cytotoxicity and lower penetration efficiency. Alternatively, these CPPs may be able to rapidly cross the lipid bilayer without damaging the cell membrane and kill bacteria by inhibiting intracellular functions.55 In contrast, three CPPs with high penetration efficiency, that is, (13) R9-TAT, (35) pAntpHD (Pro50), and (7) (KH)9, showed weaker cytotoxic effects (Figure 9). When applied at the effective concentrations, these CPPs may be internalized into E. coli DH5α cells without damaging the cell membrane, which is highly consistent with the observed localization of the CPPs in the E. coli cytoplasm (Figure S4). The intracellular localization of the TAMRA-labeled CPPs was distinguished using FM4-64 and CLSM. The results show that (13) R9-TAT displayed the highest penetration efficiency and the greatest degree of localization within the E. coli cytoplasm. On the basis of the overall results, we conclude that (13) R9-TAT is the most effective CPP against E. coli DH5α in the current CPP library. The structure−function relationship of CPP is important and essential to design highly efficient CPPs. The secondary structures of the CPPs in the library were characterized and reported previously.33 Although we have investigated the correlation between the secondary structures and cell penetrating efficiencies of the CPPs in the current study, there was no obvious correlation between the secondary structures and cell penetrating efficiency. Furthermore, the effect of the secondary structure on the cytotoxicity of CPP against E. coli was not recognized. Arg-rich peptides have been known to exhibit high penetration efficiency without significant cytotoxicity in eukaryotic mammalian cells.2,56,57 Arg residues is critical for the penetration of cationic peptides into eukaryotic mammalian cells, whereas Lys residues is not.50,58 In the case of eukaryotic plant cells, such a difference between Arg and Lys was not recognized obviously, according to a previous study.33 In this study using bacteria, namely, prokaryotic cells, (13) R9TAT, which is an Arg-rich CPP, is the most effective CPP. On the other hand, the lipid compositions of plasma membranes
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CONCLUSIONS We conducted several experiments, including FS and CLSM, to identify the best CPP for E. coli DH5α in terms of high penetration efficiency and low cytotoxicity. The results of the CLSM experiment with the double staining illustrate that the CPPs with the higher penetration efficiency entered the E. coli cells and accumulated in the cytoplasm. On the basis of the overall results, we determined that the cationic CPP R9-TAT is the best CPP for E. coli DH5α. The CPPs evaluated in this study can be used as carriers to transport therapeutic molecules, drugs, proteins, and genes into bacteria. Furthermore, we plan to construct novel functional molecules by creating fusion peptides in which CPPs are fused with other functional peptides.
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MATERIALS AND METHODS CPP Library. A CPP library containing 55 CPPs was previously constructed.33 The CPPs in the library are listed in Table S1. The CPPs were synthesized using standard 9fluorenylmethoxycarbonyl solid-phase peptide synthesis60 and included CPPs containing Lys residues modified with TAMRA via the Lys side chain amine at the C-terminus. Each CPP was purified by high-performance liquid chromatography at 25 °C. The molecular weights of the CPPs were confirmed by matrixassisted laser desorption/ionization-time-of-flight mass spectrometry. Because of their poor water solubility, (18) IX, (34) 2× ppTG1, and (50) G53-4 were dissolved in DMSO before addition to the culture medium (final DMSO concentration: 2.5%). Preparation of E. coli DH5α Chemically Competent Cells. The chemically competent cells were prepared by a previously reported procedure with minor modifications.61 Five milliliters of fresh overnight culture was diluted in 100 mL of LB growth medium. The cells were grown at 37 °C with shaking at 200 rpm to an OD600 of 0.7 (the OD600 was never higher than 0.8, except for where noted). The cells were harvested by centrifugation in a GSA rotor for 10 min at 5000 rpm, resuspended in 8 mL of 50 mM CaCl2, and collected by centrifugation for 10 min at 5000 rpm at 4 °C. Then, the washes and centrifugation were repeated using 8 mL of 50 mM CaCl2. After the decantation of the supernatant, the cells were resuspended in 4 mL of 50 mM CaCl2 containing 15% glycerol, and the mixture was incubated for 2 h on ice. The cells were aliquoted into microfuge tubes (100−200 μL/tube), frozen quickly in liquid N2, and stored at −80 °C until use. FS Experiments. The fluorescence intensity of the CPPs was analyzed using a SpectraMax M3 fluorimeter (Molecular Devices, San Jose, CA, USA) at an excitation wavelength of 544 nm, an emission wavelength of 590 nm, and an acquisition duration of 0.2 s/0.5 nm. A calibration curve of the fluorescence intensity was prepared by serial dilution of 0 to 5 μg CPPs (0.1 μg/μL) in 50 μL of Milli-Q water in a 96-well plate (Figure S1). The cell-associated CPP fluorescence intensity was measured after the addition of 5 μg of CPPs (0.1 μg/μL) to E. coli DH5α at a density of 5 × 107 cells/50 16496
DOI: 10.1021/acsomega.8b02348 ACS Omega 2018, 3, 16489−16499
ACS Omega
Article
μL. The E. coli−CPP complexes were incubated for 30 min at room temperature. Then, the complexes were washed three times with Milli-Q water and collected by centrifugation at 10 000 rpm for 10 min. Finally, the complexes were suspended in 50 μL Milli-Q water, and the fluorescence intensity was measured by FS. The fluorescence intensity of the samples was compared with the calibration curve to quantify the bacteria cell-associated CPPs (eq 1). For each experimental condition, we used duplicate wells; all experiments were repeated independently at least five times as indicated. Cell‐associated CPP intensity (au) added CPPs − free untrapped CPPs = × 100 added CPPs
CLSM Imaging of CPP Localization. The CPP localization within the bacterial cells was determined by double staining with TAMRA-labeled CPPs and FM4-64, which is a membrane-staining dye.43 TAMRA-labeled CPPs at 0.1 μg/μL were added to E. coli DH5α at a density of 5 × 107 cells/50 μL. After a 30 min incubation at RT, the cells were washed with Milli-Q water to remove the extracellular CPPs, and 0.2 μM FM4-64 was added. After further incubation for 30 min at RT, the cells were washed with Milli-Q water to remove the extracellular CPPs. Then, the samples were mounted on glass slides with cover slips, and the presence of a single layer of bacterial cells was confirmed (Figures 7 and S4). The TAMRA-labeled CPPs were visualized at an excitation wavelength of 547 nm and an emission wavelength of 590 nm. FM4-64 was visualized at excitation wavelengths of 488− 515 nm and emission wavelengths of 640−665 nm. Then, the localization of the CPPs in the E. coli cells was observed by LSM 880 (Carl Zeiss, Germany) with a C Plan-Apochromat 63×/1.4 Oil DIC UV−Vis−IR M27 objective at an excitation wavelength of 488 nm (Argon) for FM4-64 (emission at 640− 730 nm), and 561 nm (DPSS 561-10) for 5-TAMURA-labeled peptide (emission at 560−590 nm). We set the detection pinhole 1.0 times of the diameter of the Airy Disk (1 airy unit) in LSM880. The thickness of the optical sections is less than 0.8 μm. All the images were recorded digitally in a 2048 × 2048 pixel format. The internalization pattern of the CPPs was analyzed using ImageJ (Fiji) based on the RGB profiler and 3D image structure (Figures 7 and S4).62 E. coli DH5α Cell viability. The E. coli cell viability was evaluated by double staining with TAMRA-labeled CPPs and EB, which is a dye that binds only dead cells. As a positive control, suspensions of E. coli DH5α at a density of 5 × 107 cells/50 μL were treated with 70% ethanol for 30 min to kill the cells; then, 0.25% (v/v) EB44 was added, the samples were incubated for 30 min, and the cells were washed three times. As a negative control, suspensions of E. coli DH5α at a density of 5 × 107 live cells/50 μL were mixed with 0.25% (v/v) EB and washed three times. For the experimental samples, suspensions of E. coli DH5α at a density of 5 × 107 cells/50 μL were stained with CPPs at 0.1 μg/μL for 30 min. The complexes were washed with Milli-Q water and incubated with 0.25% (v/v) EB for 30 min. Then, the samples were mounted on glass slides with cover slips, and the presence of a single layer of bacterial cells was confirmed (Figure S5). The cells were observed under a LSM880 (Zeiss) microscope with a 63× oil immersion objective. The entire images were recorded digitally in a 2048 × 2048 pixel format. The images were analyzed using ImageJ (Fiji).62 The MIC of the CPPs against E. coli DH5α in the range of 0.01−20 μg/μL was determined using EB (Figure S6). Finally, the dose responses were measured at 5 μg/μL CPPs against E. coli DH5α with EB using the procedure described above. Statistical Analysis. The data are presented as the mean ± standard deviation (±SD). Statistical Package for the Social Sciences (SPSS) software version 22 (IBM Corp. Released 22.0.0.0) was used for all analyses. All data were subjected to a two-way analysis using ANOVA with the Tukey post hoc test. A p-value
