TY - JOUR
AB - Abstract Objectives The worldwide increase in antibiotic-resistant bacteria is a growing threat to public health. Antimicrobial peptides (AMPs) are potentially effective alternatives to conventional antibiotics. We therefore tested analogues of the AMP mBjAMP1 from Branchiostoma japonicum, which we produced by adding and/or replacing amino acids to increase antimicrobial activity against Gram-negative bacteria. Methods We compared the antimicrobial activities of mBjAMP1 analogues against Gram-negative bacteria reference strains and 52 strains of Klebsiella pneumoniae isolated from patients. Antibiofilm activity and cytotoxicity were evaluated, and the mechanisms of action were then studied. Results Analogue peptides exhibited greater antimicrobial and antibiofilm activities than mBjAMP1. In particular, the analogue IARR-Anal10 displayed not only the greatest antimicrobial and antibiofilm activities, but also no toxicity against human red blood cells or other mammalian cells. IARR-Anal10 had little or no effect on bacterial outer membrane permeability, membrane polarization or membrane integrity. Instead, it appears IARR-Anal10 binds bacterial DNA, as evidenced in DNA gel retardation assays. Thus, IARR-Anal10 likely kills bacteria through an intracellular mechanism. We also confirmed that IARR-Anal10 suppresses the virulence of K. pneumoniae to a degree similar to tigecycline, used to treat carbapenem-resistant Enterobacteriaceae infections. Notably, IARR-Anal10 did not induce development of resistance by K. pneumoniae, though both meropenem and tigecycline did so within a short time. Conclusions These results suggest that IARR-Anal10 is a promising agent for treating infections caused by bacteria resistant to tigecycline and meropenem. Introduction Klebsiella pneumoniae is a highly pathogenic bacterium that is of particular interest because of the large number of strains that have developed antibiotic resistance. The increase in MDR in K. pneumoniae, which can express ESBLs or AmpC β-lactamases, has led to widespread use of carbapenem antibiotics.1 As a result, however, observations of K. pneumoniae that produce carbapenemases, which are β-lactamases capable of hydrolysing carbapenems, are increasing.2 As a last resort, tigecycline, the first member of the glycylcycline class of antibiotics, is being used to overcome carbapenem-resistant K. pneumoniae infection.3 However, tigecycline-resistant K. pneumoniae have now been detected in many countries.4 Antimicrobial peptides (AMPs) have been used as alternatives to conventional antibiotics. AMPs are key components of the innate immune system and represent the first line of defence against infectious pathogens.5 AMPs commonly have broad-spectrum activity against bacteria, fungi, cancer cells and viruses. The primary mechanism of AMPs showing major antibacterial activity involves interfering with bacterial membranes in several ways.6 However, some AMPs cross the bacterial membrane to target intracellular components, including DNA, RNA and proteins present in the bacterial cytoplasm. BjAMP1 is an AMP purified from Branchiostoma japonicum, commonly known as the lancelet or amphioxus.7 The purified protein was composed of 97 amino acids, but analyses using Peptide Cutter and CAMP enabled discovery of the mature form, mBjAMP1, consisting of 21 amino acids. Notably, while mBjAMP1 has antibacterial activity, it is not toxic to mammalian cells.8 In the present study, we designed mBjAMP1 analogues by adding the amino acid sequence IARR to the peptide’s N-terminus. Comparison of their antimicrobial activities showed that IARR-mBjAMP1 performed better than did mBjAMP1. We then designed 10 additional analogues based on the IARR-mBjAMP1 model to further enhance the peptide’s antimicrobial activity. Among these analogues, IARR-Anal10 exhibited potent antibacterial activity, especially against K. pneumoniae. We then examined the antimicrobial and antibiofilm activities of IARR-Anal10 against MDR K. pneumoniae, as well as its cytotoxic activities against MRC-5 cells derived from human lung tissue. The mechanisms driving the antimicrobial actions of IARR-mBjAMP1 and IARR-Anal10 against K. pneumoniae were examined in membrane-related experiments and DNA binding assays. Also examined were their suppressive action against K. pneumoniae virulence and the development of resistance. Our findings suggest the peptides designed in this study have the potential to serve as effective treatments against infections caused by MDR K. pneumoniae. Materials and methods Peptide synthesis and design The peptides used in this study were synthesized as described previously.9 The synthesis and molecular weights of the peptides were analysed using a reversed-phase HPLC system fitted with a C18 column and MALDI-TOF MS (Figures S1 and S2, available as Supplementary data at JAC Online). We produced the analogue peptides by substituting amino acids in IARR-mBjAMP1. Because we expected that IARR-mBjAMP1 was structurally more stable than mBjAMP1, three-dimensional structural projections of mBjAMP1 and IARR-mBjAMP1 were created using Mobyle@RPBS (http://mobyle.rpbs.univ-paris-diderot.fr/cgi-bin/portal.py#welcome) (Figure S3). By replacing amino acids, we produced 10 IARR-mBjAMP1 derivatives with the aim of improving cationicity and hydrophobicity. Microorganisms Pseudomonas aeruginosa ATCC 27853 was obtained from the ATCC. Acinetobacter baumannii KCTC 2508 and K. pneumoniae KCTC 2208 were from the Korea Collection for Type Cultures (KCTC). Other K. pneumoniae strains were antibiotic-resistant bacteria isolated from patients treated at the Asan and Eulji Medical Centers. Circular dichroism (CD) spectroscopy The structural features of peptides in various solutions were evaluated as previously described.9 Antimicrobial activity tests Antimicrobial activity assays to determine MICs were performed using the broth dilution method.10 Briefly, Gram-negative, antibiotic-resistant K. pneumoniae strains were cultured in suitable culture medium at 37°C. Each strain was diluted in appropriate medium to a concentration of 2 × 105 cfu/mL. Peptides and antibiotics were diluted to concentrations ranging from 0.06 to 128 μΜ in 10 mM sodium phosphate buffer (pH 7.2) in 96-well plates. The prepared bacteria were then added to the 96-well plates and incubated for 16–24 h at 37°C. Growth was determined by measuring the absorbance at 600 nm using a microplate reader. Preparation of large unilamellar vesicles (LUVs) for the aggregation assay LUVs were prepared using the freeze–thaw method. Briefly, the following lipid mixtures were prepared in chloroform: (i) phosphatidylcholine (PC):cholesterol (CH) (2:1 w/w); and (ii) PC:CH:sphingomyelin (SM) (1:1:1 w/w/w). The prepared mixtures were dried under argon. To completely remove the chloroform, the mixtures were lyophilized for 24 h, after which the dried lipid films were resuspended in 2.6 mL of PBS by vortexing for 30 min. The resulting suspensions were frozen and thawed nine times using liquid nitrogen and a water bath at 50°C, alternately, and then extruded >20 times through 0.2 μm polycarbonate membranes. The lipid concentration was confirmed using a standard phosphate assay.11 Haemolysis The haemolytic activity of peptides was assayed as previously described.12 Cytotoxicity We studied the cytotoxicity of the peptides using an IncuCyte® Cytotoxicity Assay (Essen Bioscience) with MRC-5 cells. The cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin at 37°C with 5% CO2. After treating the cells with peptides, the cytotoxicity-related fluorescent signal was monitored for 24 h. Images of the cytotoxicity-related fluorescence were obtained. Biofilm inhibition assays To assess biofilm inhibition by the peptides, 90 μL (5 × 105 cfu/mL) of carbapenem-resistant K. pneumoniae bacterial suspension in nutrient broth and 10 μL of peptide or antibiotic at concentrations ranging from 1 to 64 μM were then added into the plate, and the mixtures were incubated for 24 h at 37°C. The supernatant was then carefully discarded, and the biofilms formed on the plates were fixed by adding 100% methanol for 10 min. The methanol was then gently removed, and the biofilms were stained with 0.1% crystal violet for 30 min. The plates were then washed with water until the control wells appeared colourless. Finally, the stained biofilms were dissolved in 100% ethanol and the absorbance was measured at 595 nm using a Versa-Max microplate ELISA reader (Molecular Devices, Sunnyvale, CA, USA). Percentage biofilm mass was calculated using the equation (A595 of the treated biofilm/A595 of the untreated biofilm)×100. The minimum biofilm inhibitory concentration (MBIC) was set at the lowest concentration that inhibited biofilm by 50%.13 Determination of bacterial viability within the biofilms After staining biofilms exposed to peptides or antibiotics, they were visualized using an IX71 inverted fluorescence microscope (Olympus, Tokyo, Japan). Briefly, 90 μL of carbapenem-resistant K. pneumoniae suspension in nutrient broth and 10 μL of peptide or antibiotic at concentrations ranging from 1 to 64 μΜ were incubated in tissue culture plastic plates for 24 h at 37°C. After incubation, the biofilms were stained with 100 μL of live cell dye (SYTO 9) for 30 min in the dark. Biofilm eradication assay To form biofilms, 100 μL aliquots of carbapenem-resistant K. pneumoniae (ASKP 0002) (5 × 105 cfu/mL) suspension in nutrient broth were inoculated into 96-well flat-bottomed polystyrene culture plates and grown for 48 h at 37°C. The culture medium was then carefully removed, and the wells were washed with PBS to remove planktonic cells, leaving only the biofilm. Thereafter, peptides or antibiotics at 200 μM in nutrient broth were added every 24 h for 3 days. For confocal microscopic observation, the medium was carefully removed, and the biofilms were washed with PBS and stained with SYTO 9 fluorescent dye for 30 min in the dark. Images were then obtained using an EVOS FL Auto 2 imaging system (Invitrogen).14,15 Outer membrane permeability assay Changes in outer membrane permeability caused by the peptides were measured using a 1-N-phenylnaphthylamine (NPN) uptake assay.16 Membrane depolarization K. pneumoniae (KCTC 2208) cells were resuspended in 20 mM glucose, 5 mM HEPES (pH 7.2) and 100 mM KCl. The membrane-potential-sensitive dye DiSC3(5) (Sigma–Aldrich, St Louis, MO, USA) was added, and the mixture was incubated until the fluorescence had stabilized. The peptides were subsequently added to the mixture, and increases in fluorescence indicating membrane depolarization were measured for 30 min using an excitation wavelength of 622 nm and an emission wavelength of 670 nm.17 SYTOX green uptake assay K. pneumoniae (KCTC 2208) cells were washed and resuspended in 10 mM sodium phosphate buffer (pH 7.2). The bacteria were incubated with SYTOX green for 15 min in the dark, after which they were mixed with peptides at various concentrations, and increases in fluorescence were measured (excitation at 485 nm and emission at 520 nm).17 Calcein leakage from liposomes We prepared LUVs composed of phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL) at a 6:0.9:1 w/w/w/ratio as described above, except that the dry lipid films were resuspended in PBS (pH 7.2) containing 70 mM calcein. Gel filtration chromatography on a Sephadex G-50 column was used to separate the free calcein from calcein entrapped in LUVs. Thereafter, the LUVs with entrapped calcein were mixed with peptides at various ratios, and the fluorescence from the leaked calcein was measured using a Spectramax M3 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA), with excitation at 480 nm and emission at 520 nm. Flow cytometry K. pneumoniae (KCTC 2208) cells were washed with 10 mM PBS (pH 7.4) and diluted to an OD600 of 0.2 in 10 mM PBS. The bacterial suspensions were incubated with peptides or 10 mM PBS (negative control) for 2 h at 37°C. After incubation, propidium iodide (0.1 mg/mL) was added, and the suspension was left to incubate for 30 min at 4°C. The bacteria were then washed with 10 mM PBS to remove any unbound dye. Data were collected using a CytoFLEX flow cytometer (Beckman Coulter, Brea, CA, USA). DNA binding assay Samples (250 ng) of plasmid DNA (pRSETA) were mixed with increasing concentrations of peptides in a buffer containing 10 mM Tris–HCl (pH 8.0), 1 mM EDTA, 5% glucose, 20 mM KCl and 50 μg/mL BSA. The mixtures were then incubated for 10 min at 37°C, after which DNA sample buffer was added to each vial, and aliquots were used to perform electrophoresis using agarose gels (1% agarose, 100 V) in 0.5 × Tris/acetate/EDTA (TAE) buffer. The gels were then stained with ethidium bromide, and the DNA bands were visualized using UV illumination with a Bio-Rad gel documentation system (Hercules, CA, USA). Confocal laser scanning microscopy To confirm the cellular distributions of IARR-Anal10, K. pneumoniae (KCTC 2208) cells were incubated in the presence of tetramethyl rhodamine (TAMRA)-labelled peptide and observed using a confocal laser scanning microscope. Bacteria were cultured in nutrient broth to a concentration of 2 × 107 cfu/mL, after which TAMRA-labelled peptides at their respective MICs were added, and the cultures were incubated for 1 h. The mixture was then centrifuged at 4000 rpm for 5 min and washed three times with PBS buffer. Localization of the TAMRA-labelled peptides was confirmed using a confocal microscope (Zeiss LSM 510 Meta; Carl Zeiss). Effects of peptides and antibiotics on expression of virulence genes K. pneumoniae (ASKP 00002) cells were incubated for 18 h in the presence of a peptide or antibiotic. Total RNA was then isolated using TRIzol reagent (Ambion™) and quantified using a spectrophotometer (NanoDrop 2000, Thermo Fisher Scientific). cDNA was synthesized using TOPscript™ RT DryMIX (Enzynomics) and used to amplify the internal standard (16S rRNA) used for normalization, the siderophore gene ybtS, the pili gene mrkA, the capsular gene galF and the K. pneumoniae carbapenemase-2 gene (KPC-2) using an Applied Biosystems 7500 Real-Time PCR system. The amplification parameters for ybtS, galF and mrkA were as follows: denaturation at 95°C for 2 min, followed by 45 cycles of 95°C for 10 s, 57°C for 20 s and 72°C for 30 s. For KPC-2, the following parameters were applied: denaturation at 95°C for 15 min, followed by 45 cycles of 95°C for 10 s, 55°C for 30 s and 72°C for 30 s. The relative expression levels were then compared with those obtained in untreated bacterial samples using the following equation: gene expression (%) = (treated sample/untreated sample) × 100. Peptide and antibiotic resistance assay We modified the resistance assay from previously described methods.18 Briefly, to obtain resistant cells, the MICs of IARR-Anal10, tigecycline and meropenem for K. pneumoniae (KCTC 2208) were determined as described above. K. pneumoniae cells were then inoculated into nutrient broth containing 0.5 × MIC of a peptide or antibiotics for 16 h. The inoculum was measured for MIC and again incubated with 0.5 × MIC for 16 h. This process was repeated up to 30 times. Results and discussion AMPs have been studied as an alternative to conventional antibiotics. After the 97 amino acid protein BjAMP1 was isolated from B. japonicum, Peptide Cutter and CAMP were used to identify the mature AMP, mBjAMP1, composed of 21 amino acids, located at the C-terminus of BjAMP1.8 We anticipated that a more stable α-helical structure would be produced by adding four amino acids (IARR) to the C-terminus, at the front of mBjAMP1. Because the helicity of AMPs influences their antimicrobial activity,19 we expected that the increased helicity of IARR-mBjAMP1 would result in greater antimicrobial activity against Gram-negative bacteria than was shown by mBjAMP1. Ten analogue peptides, based on IARR-mBjAMP1, were then designed by replacing various residues with alanine (A), thereby increasing amphipathicity as well as activity.20 The amino acid sequences, molecular weights, hydrophobicity and retention times of the peptides are summarized in Table S1. Lysine (K) is a cationic residue that enables electrostatic interactions with negatively charged bacterial membranes. The net charge values of the analogue peptides, which ranged from +8 to +11, are all greater than that of mBjAMP1 (+6). The hydrophobicity of the analogue peptides ranged between 0.165 and 0.306, which is also greater than that of the parent peptide. AMPs act either through membrane disruption or binding to intracellular targets. As controls, therefore, we used melittin, which is known to form toroidal pores, as a representative lytic peptide, and buforin 2, which inhibits bacterial function by binding to its DNA after penetrating the cell membrane, as an intracellularly acting agent.21,22 The antimicrobial activities of peptides and antibiotics against Gram-negative and antibiotic-resistant K. pneumoniae are shown in Table S2. mBjAMP1 had broad-spectrum antimicrobial activity against A. baumannii, P. aeruginosa and K. pneumoniae reference strains, with MICs ranging from 16 to 32 μΜ. By contrast, the MICs of IARR-mBjAMP1 analogues ranged from 8 to 16 μΜ for Gram-negative bacteria. Moreover, IARR-Anal9 and IARR-Anal10 were the most active against K. pneumoniae. We confirmed their antimicrobial activity against 52 strains of K. pneumoniae isolated from hospital patients (Tables S3 and S4). IARR-Anal10 showed greater antimicrobial activity than the parent peptide and the other analogues against most K. pneumoniae strains. Notably, for several strains, the peptides had lower MICs than meropenem (Table S5). Thus, IARR-Anal10 exhibits potent antimicrobial activity against K. pneumoniae. The secondary structure of AMPs includes α-helixes, β-sheets and loops. When we used CD spectroscopy to determine the secondary structures of IARR-mBjAMP1 and IARR-Anal10,23 we found that both assumed a random coil structure in 10 mM sodium phosphate (pH 7.4), mimicking an aqueous environment (Figure S4a). On the other hand, both peptides displayed an α-helical structure in 30 mM SDS, which mimics the negatively charged bacterial surface, and in trifluoroethanol (TFE), which mimics a hydrophobic environment and can support and induce α-helical structure in peptides (Figure S4b and c).24,25 The ability of an AMP to neutralize LPS, a well-known endotoxin, is paramount. Because AMPs are cationic, they interact with the anionic LPS, illustrating a way in which the amphipathic properties of the α-helical structures IARR-mBjAMP1 and IARR-Anal10 underlie their antimicrobial activities (Figure S4d).26,27 Liposome aggregation is a phenomenon related to the interaction between peptides and lipids.28 Eukaryotic cell membranes are composed of PC and SM, which are electrically neutral. Bacterial membranes, by contrast, are composed of negatively charged phospholipids such as PG and CL.29 As a result of this charge difference, peptides can be constructed to selectively act on prokaryotic and bacterial membranes. To assess the interaction between lipids and IARR-mBjAMP1 or IARR-Anal10, we measured the absorbance changes resulting from the aggregation of the peptides and liposomes. IARR-mBjAMP1 and IARR-Anal10 were separately mixed with liposomes at various peptide/liposome ratios. PC:CH was used to mimic human red blood cell (hRBC) membranes, whereas PC:CH:SM was used to mimic other mammalian membranes. Both IARR-mBjAMP1 and IARR-Anal10 induced much less aggregation of both types of liposome than melittin, which served as a positive control (Figure 1a). To evaluate the cytotoxicity of the peptides, we assessed their haemolytic activity by measuring the haemoglobin released from hRBCs upon exposure to each peptide, which causes the serum or plasma sample to appear pale red or cherry red in colour.30 The concentration–response curves in Figure 1(b) show that melittin, the positive control, induced haemolysis in 83% of cells at 1.56 μΜ. By contrast, IARR-mBjAMP1 and IARR-Anal10 showed haemolytic activities <5% at 200 μM. We further investigated the cytotoxicity of the peptides using MRC-5 cells derived from human lung tissue. Using IncuCyte cytotoxicity assays, which enabled us to obtain real-time, fluorescence-based measurements of cytotoxicity, we found that IARR-mBjAMP1 and IARR-Anal10 induced no increases in cell death (green fluorescence) above control after 24 h of exposure (Figure 1c and d), whereas 25 μΜ melittin quickly increased fluorescence. Figure 1. View largeDownload slide (a) Liposome aggregation assay. AMPs were mixed with LUVs composed of PC:CH:SM (1:1:1 w/w; mimicking most mammalian membranes) and PC:CH (2: 1 w/w; mimicking hRBC membranes) at different peptide:liposome ratios. Aggregation was monitored based on the absorbance at 405 nm. (b) Haemolytic activities of AMPs and antibiotics. The y-axis shows percentage haemolysis of hRBCs calculated as follows: percentage haemolysis = [(A414 in peptide and antibiotic solutions – A414 in PBS)/(A414 in 0.1% Triton X-100 – A414 in PBS)] × 100. (c) Cytotoxicity of AMPs after 24 h of exposure. The cytotoxicity of AMPs at the indicated concentrations was measured as a function of green fluorescence. (d) Images of cells loaded with green cytotoxicity reagent before and after exposure to the indicated peptide for 24 h. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Figure 1. View largeDownload slide (a) Liposome aggregation assay. AMPs were mixed with LUVs composed of PC:CH:SM (1:1:1 w/w; mimicking most mammalian membranes) and PC:CH (2: 1 w/w; mimicking hRBC membranes) at different peptide:liposome ratios. Aggregation was monitored based on the absorbance at 405 nm. (b) Haemolytic activities of AMPs and antibiotics. The y-axis shows percentage haemolysis of hRBCs calculated as follows: percentage haemolysis = [(A414 in peptide and antibiotic solutions – A414 in PBS)/(A414 in 0.1% Triton X-100 – A414 in PBS)] × 100. (c) Cytotoxicity of AMPs after 24 h of exposure. The cytotoxicity of AMPs at the indicated concentrations was measured as a function of green fluorescence. (d) Images of cells loaded with green cytotoxicity reagent before and after exposure to the indicated peptide for 24 h. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Biofilms can lead to chronic infections and increased resistance to antibiotic treatments. We used four carbapenem-resistant K. pneumoniae strains able to form biofilms to compare the antibiofilm activities of IARR-mBjAMP1 and IARR-Anal10 with those of two conventional antibiotics, meropenem and tigecycline. Meropenem did not inhibit biofilm formation, even at high concentrations. IARR-Anal10 and tigecycline exhibited similar abilities to inhibit biofilm formation by carbapenem-resistant K. pneumoniae (Figure 2a–d). To assess the viability of the bacteria within the biofilm, live BacLight staining (SYTO 9) was used. We treated the biofilms with IARR-mBjAMP1, IARR-Anal10 or tigecycline at their MBIC, or meropenem at 64 μM. With the two peptides and tigecycline, the numbers of live bacteria within the biofilms were markedly decreased by treatment. On the other hand, none of the biofilms was measurably affected by meropenem (Figure 2e). Also evaluated were the efficacies of AMPs and antibiotics against carbapenem-resistant K. pneumoniae within established biofilms. Although we observed no decrease in biofilm after three administrations of IARR-mBjAMP1, meropenem or tigecycline at 200 μM, administration of IARR-Anal10 led to significant decreases in live cells within biofilms (Figure 2f). IARR-Anal10 thus appears to exert strong antibiofilm effects against carbapenem-resistant K. pneumoniae. Figure 2. View largeDownload slide View largeDownload slide Comparison of the antibiofilm activities of peptides and antibiotics. Dose–response curves showing the antibiofilm effects of the indicated peptides and antibiotics against carbapenem-resistant K. pneumoniae strains: (a) ASKP 00001, (b) ASKP 00002, (c) ASKP 00003 and (d) ASKP 00005. Biofilm mass was determined by staining with crystal violet and measuring absorbance at 595 nm. (e) Fluorescence microscopic analyses of biofilms formed by K. pneumoniae and the effects of treatment with the indicated peptides and antibiotics. Live cells were stained with SYTO 9. MBIC values were determined as described in the Materials and methods section. ASKP 00001 (IARR-mBjAMP1, 32 μΜ; IARR-Anal10, 4 μΜ; tigecycline, 1 μΜ), ASKP 00002 (IARR-mBjAMP1, 16 μΜ; IARR-Anal10, 8 μΜ; tigecycline, 2 μΜ), ASKP 00003 (IARR-mBjAMP1, 64 μΜ; IARR-Anal10, 8 μΜ; tigecycline 2 μΜ), ASKP 00005 (IARR-mBjAMP1, 32 μΜ; IARR-Anal10, 4 μΜ; tigecycline, 1 μΜ). (f) Effect of AMPs and antibiotics on established ASKP 00002 biofilms. Images show the results of adding AMPs or antibiotics at 200 μΜ to established biofilms. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Figure 2. View largeDownload slide View largeDownload slide Comparison of the antibiofilm activities of peptides and antibiotics. Dose–response curves showing the antibiofilm effects of the indicated peptides and antibiotics against carbapenem-resistant K. pneumoniae strains: (a) ASKP 00001, (b) ASKP 00002, (c) ASKP 00003 and (d) ASKP 00005. Biofilm mass was determined by staining with crystal violet and measuring absorbance at 595 nm. (e) Fluorescence microscopic analyses of biofilms formed by K. pneumoniae and the effects of treatment with the indicated peptides and antibiotics. Live cells were stained with SYTO 9. MBIC values were determined as described in the Materials and methods section. ASKP 00001 (IARR-mBjAMP1, 32 μΜ; IARR-Anal10, 4 μΜ; tigecycline, 1 μΜ), ASKP 00002 (IARR-mBjAMP1, 16 μΜ; IARR-Anal10, 8 μΜ; tigecycline, 2 μΜ), ASKP 00003 (IARR-mBjAMP1, 64 μΜ; IARR-Anal10, 8 μΜ; tigecycline 2 μΜ), ASKP 00005 (IARR-mBjAMP1, 32 μΜ; IARR-Anal10, 4 μΜ; tigecycline, 1 μΜ). (f) Effect of AMPs and antibiotics on established ASKP 00002 biofilms. Images show the results of adding AMPs or antibiotics at 200 μΜ to established biofilms. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. To gain insight into the mechanism underlying the antimicrobial actions of IARR-mBjAMP1 and IARR-Anal10, we assessed the membrane permeabilization capacities of the peptides using calcein entrapped within LUVs composed of K. pneumoniae-like membranes (PE:PG:CL). Like buforin 2, IARR-mBjAMP1 and IARR-Anal10 rarely induced calcein leakage (Figure 3a). This suggests IARR-mBjAMP1 and IARR-Anal10 act intracellularly rather than at the cell membrane. Figure 3. View largeDownload slide AMP mechanism of action. (a) Peptide-induced leakage of calcein from LUVs. AMPs were mixed with calcein-entrapping LUVs composed of PE:PG:CL 6:0.9:1 w/w/w (mimicking K. pneumoniae membrane) at different peptide/LUV ratios. (b) Effect of AMPs on K. pneumoniae outer membrane permeability. K. pneumoniae cells were exposed to the indicated concentrations of IARR-mBjAMP1 and IARR-Anal10, as described in the Materials and methods section. Time-dependent NPN uptake was then measured by monitoring its fluorescence (excitation at 350 nm and emission at 420 nm). (c) Effect of AMPs on K. pneumoniae cytoplasmic membrane polarization. After loading suspensions of K. pneumoniae cells with the membrane-potential-sensitive dye DiSC3(5), they were exposed to the indicated concentration of IARR-mBjAMP1 or IARR-Anal10. Time-dependent changes in membrane potential were then monitored as a function of fluorescence (excitation at 622 nm and emission at 670 nm). Buffer (5 mM HEPES including 20 mM glucose and 10 mM KCl) was used as the control. (d) Evaluation of K. pneumoniae membrane integrity after treatment with AMPs using SYTOX green. Bacterial cells (2 × 107 cfu/mL) loaded with SYTOX green were exposed to the indicated concentrations of IARR-mBjAMP1 or IARR-Anal10, and the time-dependent changes in fluorescence (excitation at 485 nm and emission at 520 nm) were monitored. (e) Flow cytometric evaluation of bacterial membrane integrity using propidium iodide. K. pneumoniae cells were exposed to IARR-mBjAMP1, IARR-Anal10, buforin 2 or melittin (all at 1 × MIC) for 2 h. (f) Localization of peptides on K. pneumoniae (2 × 107 cfu/mL) incubated with TAMRA-labelled IARR-Anal10 (0.5 × MIC) for 1 h at 37°C. The bacteria were stained with SYTO 9. (g) DNA gel retardation assays assessing peptide binding to DNA. Plasmid DNA at a fixed concentration was incubated with various concentrations of IARR-mBjAMP1, IARR-Anal10 or buforin 2 before electrophoresis in a 1% agarose gel. The lanes represent the following peptide:DNA ratios: lane 1, only DNA; lane 2, 0.25:1; lane 3, 0.5:1; lane 4, 0.75:1; lane 5, 1:1; lane 6, 1.5:1; lane 7, 2:1; and lane 8, 2.5:1. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. Figure 3. View largeDownload slide AMP mechanism of action. (a) Peptide-induced leakage of calcein from LUVs. AMPs were mixed with calcein-entrapping LUVs composed of PE:PG:CL 6:0.9:1 w/w/w (mimicking K. pneumoniae membrane) at different peptide/LUV ratios. (b) Effect of AMPs on K. pneumoniae outer membrane permeability. K. pneumoniae cells were exposed to the indicated concentrations of IARR-mBjAMP1 and IARR-Anal10, as described in the Materials and methods section. Time-dependent NPN uptake was then measured by monitoring its fluorescence (excitation at 350 nm and emission at 420 nm). (c) Effect of AMPs on K. pneumoniae cytoplasmic membrane polarization. After loading suspensions of K. pneumoniae cells with the membrane-potential-sensitive dye DiSC3(5), they were exposed to the indicated concentration of IARR-mBjAMP1 or IARR-Anal10. Time-dependent changes in membrane potential were then monitored as a function of fluorescence (excitation at 622 nm and emission at 670 nm). Buffer (5 mM HEPES including 20 mM glucose and 10 mM KCl) was used as the control. (d) Evaluation of K. pneumoniae membrane integrity after treatment with AMPs using SYTOX green. Bacterial cells (2 × 107 cfu/mL) loaded with SYTOX green were exposed to the indicated concentrations of IARR-mBjAMP1 or IARR-Anal10, and the time-dependent changes in fluorescence (excitation at 485 nm and emission at 520 nm) were monitored. (e) Flow cytometric evaluation of bacterial membrane integrity using propidium iodide. K. pneumoniae cells were exposed to IARR-mBjAMP1, IARR-Anal10, buforin 2 or melittin (all at 1 × MIC) for 2 h. (f) Localization of peptides on K. pneumoniae (2 × 107 cfu/mL) incubated with TAMRA-labelled IARR-Anal10 (0.5 × MIC) for 1 h at 37°C. The bacteria were stained with SYTO 9. (g) DNA gel retardation assays assessing peptide binding to DNA. Plasmid DNA at a fixed concentration was incubated with various concentrations of IARR-mBjAMP1, IARR-Anal10 or buforin 2 before electrophoresis in a 1% agarose gel. The lanes represent the following peptide:DNA ratios: lane 1, only DNA; lane 2, 0.25:1; lane 3, 0.5:1; lane 4, 0.75:1; lane 5, 1:1; lane 6, 1.5:1; lane 7, 2:1; and lane 8, 2.5:1. This figure appears in colour in the online version of JAC and in black and white in the print version of JAC. To further explore the peptides’ actions at the bacterial cell membrane, we examined their effect on outer membrane permeability using NPN uptake assays. NPN is a hydrophobic fluorescent probe that exhibits little fluorescence in the extracellular environment, but its fluorescence increases when the outer membrane is disrupted, giving it access to a hydrophobic environment.31 IARR-mBjAMP1 and IARR-Anal10 had little ability to increase outer membrane permeability (Figure 3b). The two peptides also did not induce membrane depolarization, as measured using the membrane-potential-sensitive dye DISC3(5), which accumulates in the lipid bilayer, causing self-quenching, and is released upon disruption of the cytoplasmic membrane, resulting in an increase in fluorescence (Figure 3c).32 The integrity of the bacterial membrane in the presence of IARR-mBjAMP1 and IARR-Anal10 was further confirmed using SYTOX green and propidium iodide. These dyes do not penetrate the intact bacterial membrane; however, when the membrane is compromised by antimicrobial agents, they enter the cell and bind to DNA, resulting in an increase in fluorescence intensity. SYTOX green fluorescence intensity was nearly unchanged when IARR-mBjAMP1 and IARR-Anal10 were added to K. pneumoniae (Figure 3d). This finding was confirmed by flow cytometry analysis using propidium iodide. Among control cells without peptides, the proportion of cells taking up propidium iodide was 14.88%. Among cells treated with IARR-mBjAMP1, IARR-Anal10 or buforin 2 at their MICs, propidium iodide was taken up by 15.59%, 12.96% and 23.99% of cells, respectively. By contrast, treatment with melittin at its MIC dramatically increased the cell fraction taking up propidium iodide to 54.83% (Figure 3e). Given that IARR-mBjAMP1 and IARR-Anal10 have little effect on bacterial membranes, we examined their intracellular effects. Confocal laser scanning microscopic examination confirmed that IARR-Anal10 penetrates the cell membrane of K. pneumoniae and localizes within the cytoplasm (Figure 3f). To determine whether the intracellular peptides target DNA or RNA, we assessed their ability to retard DNA migration during gel electrophoresis. Various concentrations of IARR-mBjAMP1, IARR-Anal10 or buforin 2 were mixed with a fixed concentration of plasmid DNA, after which the mixtures were electrophoresed on an agarose gel. IARR-mBjAMP1 completely blocked gel migration of DNA at a peptide:DNA ratio of 2. Buforin 2 also inhibited DNA migration at a peptide:DNA ratio of 2, but not as strongly as IARR-mBjAMP1. IARR-Anal10 inhibited gel migration of DNA at a peptide:DNA ratio of 0.5. Thus, IARR-Anal10 appears to have greater antimicrobial activity than buforin 2 or IARR-mBjAMP1 (Figure 3g). Virulence factors from K. pneumoniae likely mediate inflammation in humans. Information on the gene characterization and primers is presented in Table S6. We confirmed the ability of IARR-Anal10 to suppress expression of representative virulence factors, such as siderophores, pili, capsular polysaccharides and carbapenemase-2. Siderophores, which are important for bacterial growth, are iron-chelating molecules. They are encoded by ybtS and secreted by K. pneumoniae, which induces immune responses and favours bacterial dissemination.33 Pili in K. pneumoniae contribute to the initial colonization leading to biofilm formation and are classified as type 1 pili (T1P) or type 3 pili (T3P). The pilus gene mrkA is directly involved in biofilm formation. Mutation of mrkA in K. pneumoniae reduces its ability to form biofilms.34 Capsular polysaccharides (CPSs) help protect K. pneumoniae against phagocytosis.35,galF, wzi and manC are located within the CPS gene cluster in K. pneumoniae.34 Carbapenem resistance is mediated by KPC-2, which inactivates carbapenems through hydrolysis. IARR-Anal10 and tigecycline down-regulated expression of virulence genes by ∼20%–50% compared with the control. Meropenem also did not inhibit most virulence genes and it also elicited a 3% increase in KPC-2 expression (Figure 4). These results suggest that IARR-Anal10 may have a suppressive effect on inflammation by inhibiting the virulence genes from K. pneumoniae. Figure 4. View largeDownload slide Effect of AMPs and antibiotics on expression of K. pneumoniae virulence genes. K. pneumoniae (ASKP 00002) at 2 × 105 cfu/mL was exposed to the indicated AMPs and antibiotics at 0.5 × MIC for 18 h at 37°C, after which gene expression levels were determined using quantitative SYBR green real-time PCR. Columns indicate the mean; bars show the SEM (n = 4). *P < 0.05, **P < 0.01 versus control (two-tailed Student’s t-test). Figure 4. View largeDownload slide Effect of AMPs and antibiotics on expression of K. pneumoniae virulence genes. K. pneumoniae (ASKP 00002) at 2 × 105 cfu/mL was exposed to the indicated AMPs and antibiotics at 0.5 × MIC for 18 h at 37°C, after which gene expression levels were determined using quantitative SYBR green real-time PCR. Columns indicate the mean; bars show the SEM (n = 4). *P < 0.05, **P < 0.01 versus control (two-tailed Student’s t-test). One of the advantages of AMPs is their low impact on bacterial resistance development. We compared the development of resistance to IARR-Anal10 with development of meropenem and tigecycline resistance (Table S7). The MIC was unaffected when K. pneumoniae cells were exposed to IARR-Anal10 at one-half its MIC for >30 passages. Treatment with meropenem at one-half its MIC led to increased resistance from the sixth passage such that the MIC was increased ∼8-fold after 30 passages. Resistance of K. pneumoniae to tigecycline developed after only two passages, and the MIC had increased ∼32-fold after 30 passages (Figure 5). These results indicate that meropenem and tigecycline induce the development of resistance in K. pneumoniae, whereas IARR-Anal10 does not. IARR-Anal10 can be considered as a promising candidate for the treatment of infections caused by K. pneumoniae. Figure 5. View largeDownload slide Treatment-induced changes in the IARR-Anal10, meropenem and tigecycline MICs for the reference strain. K. pneumoniae (2 × 105 cfu/mL) cells were inoculated in nutrient broth with the indicated agent at 0.5 × MIC for 16 h. The MICs for the inocula were then measured, after which the inocula were incubated with the agents at 0.5 × MIC for another 16 h. This process was repeated up to 30 times. Figure 5. View largeDownload slide Treatment-induced changes in the IARR-Anal10, meropenem and tigecycline MICs for the reference strain. K. pneumoniae (2 × 105 cfu/mL) cells were inoculated in nutrient broth with the indicated agent at 0.5 × MIC for 16 h. The MICs for the inocula were then measured, after which the inocula were incubated with the agents at 0.5 × MIC for another 16 h. This process was repeated up to 30 times. Acknowledgements The K. pneumoniae strains used in this study were obtained from the Eulji and Asan Medical Centers. Funding This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean Government (No. 2016R1A2A1A05005440) and a Global Research Laboratory (GRL) grant (No. NRF-2014K1A1A2064460). Transparency declarations None to declare. Supplementary data Figures S1 to S4 and Tables S1 to S7 are available as Supplementary data at JAC Online. References 1 Juan C-H , Huang Y-W , Lin Y-T et al. Risk factors, outcomes, and mechanisms of tigecycline-nonsusceptible Klebsiella pneumoniae bacteremia . Antimicrob Agents Chemother 2016 ; 60 : 7357 – 63 . Google Scholar PubMed 2 Hirsch EB , Tam VH. Detection and treatment options for Klebsiella pneumoniae carbapenemases (KPCs): an emerging cause of multidrug-resistant infection . J Antimicrob Chemother 2010 ; 65 : 1119 – 25 . Google Scholar CrossRef Search ADS PubMed 3 Pournaras S , Koumaki V , Spanakis N et al. 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TI - Antibacterial activity and mechanism of action of analogues derived from the antimicrobial peptide mBjAMP1 isolated from Branchiostoma japonicum
JF - Journal of Antimicrobial Chemotherapy
DO - 10.1093/jac/dky144
DA - 2018-04-30
UR - https://www.deepdyve.com/lp/oxford-university-press/antibacterial-activity-and-mechanism-of-action-of-analogues-derived-2PHh1Syo0D
SP - 1
EP - 2063
VL - Advance Article
IS - 8
DP - DeepDyve
ER -