Arch. Pharm. Res. DOI 10.1007/s12272-015-0600-0
RESEARCH ARTICLE
Effect of N-terminal truncation on antibacterial activity, cytotoxicity and membrane perturbation activity of Cc-CATH3 Jiraphun Jittikoon1,2 • Narumon Ngamsaithong1 • Jutarat Pimthon2,3 Opa Vajragupta2,3
•
Received: 20 November 2014 / Accepted: 3 April 2015 The Pharmaceutical Society of Korea 2015
Abstract A series of amino-terminal truncated analogues of quail antimicrobial peptide Cc-CATH3(1-29) were created and examined antibacterial activity against Grampositive bacteria, cytotoxicity against mouse fibroblast cell line, and membrane perturbation activity against various membrane models. Parent peptide Cc-CATH3(1-29) and the first four-residue truncated peptide Cc-CATH3(5-29) were active in all tested experiments. In contrast, the eightand twelve-residue truncated variants Cc-CATH3(9-29) and Cc-CATH3(13-29) appeared to have lost activities. CcCATH3(1-29) and Cc-CATH3(5-29) possessed antibacterial activity with minimum inhibitory concentrations of 2–4 and 1–2 lM, respectively. For cytotoxicity, Cc-CATH3(129) and Cc-CATH3(5-29) displayed cytotoxicity with the IC50 values of 9.33 and 4.93 lM, respectively. CcCATH3(5-29) induced greater liposome membranes disruption than Cc-CATH3(1-29) regardless of lipid type and composition. The leakage results of Cc-CATH3(1-29) share a similar trend with that in Cc-CATH3(5-29); they exhibit no preferential binding to anionic phospholipids. In conclusion, the results suggested that the first four residues at the N-terminus ‘‘RVRR’’ is not essential for presenting all test activities. In contrast, residues five to eight of
& Jiraphun Jittikoon
[email protected] 1
Department of Biochemistry, Faculty of Pharmacy, Mahidol University, 447 Sri-Ayuthaya Road, Rajathevi, Bangkok 10400, Thailand
2
Center of Excellence for Innovation in Drug Design and Discovery, Faculty of Pharmacy, Mahidol University, 447 Sri-Ayuthaya Road, Rajathevi, Bangkok 10400, Thailand
3
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Mahidol University, Bangkok 10400, Thailand
‘‘FWPL’’ are necessary as the exclusion of this short motif in Cc-CATH3(9-29) and Cc-CATH3(13-29) leads to a loss of activities. This study will be beneficial for further design and development of Cc-CATH3 to be novel antibiotic. Keywords Antimicrobial peptide Cathelicidin Peptide truncation Antibacterial Cytotoxicity Membrane perturbation
Introduction The public health worldwide has encountered a serious problem of conventional antibiotics losing their effectiveness particularly against pathogens that were once treatable but are becoming resistant. Several promising antimicrobial agents have been studied and developed to serve as potential alternatives to conventional antibiotics. Antimicrobial peptides (AMPs) are regarded as excellent candidates owing to their higher or equal potencies and broad spectrum antimicrobial activities with less possibility of resistance induction when compared to conventional antibiotic drugs (Wimley and Hristova 2011). They have increasingly been used as templates for the research and development in the field of new antibiotic drug discovery. While differing in sequence and structure, AMPs share, to a certain degree, common features such as low molecular weight, net positive charge, percent hydrophobicity, and amphipathicity. These features are believed to govern activity of AMPs (Rotem et al. 2006). Cathelicidins, which are major part of AMPs, have been identified in various species including human (e.g. human LL-37), fish, reptiles, and avians (e.g. chicken fowlicidin) (Zasloff 2002; Du¨rr et al. 2006; Xiao et al. 2006). Mammalian cathelicidins exhibit potent antimicrobial activity against a wide range of
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bacteria, fungi and enveloped viruses at micromolar concentrations (Zasloff 2002). Like most AMPs, they are thought to selectively kill microorganisms without causing toxicity to host cells (Matsuzaki 2009). The cationic side chains of cathelicidins interact with the negatively charged components which present ubiquitously in bacterial membranes. Consequently, the hydrophobic side chains perturb the lipid bilayer resulting in transient pore-formation. Avain cathelicidins appear to have captured attention as possible new, novel antimicrobial agents given their superior antibacterial potency and effectiveness than other cathelicidins that have been identified to date (van Dijk et al. 2011). Chicken cathelicidins or fowlicidins were reported to be one of the most potent cathelicidins, with minimum inhibitory concentrations (MICs) less than 2 lM against a variety of bacteria (Xiao et al. 2006). Cc-CATH3, a cathelicidin from quail (Coturnix coturnix), has recently been characterized and also proved to be a strong new AMP candidate for therapeutic development (Feng et al. 2011). Cc-CATH3(1-29), composed of 29 amino acids, manifested broad-spectrum antimicrobial activity against a variety of microorganisms including bacteria and fungi as well as some drug-resistant strains with MIC values in the range of 0.3–2.5 lM, a higher potency than those of ampicillin and kanamycin, while showing low cytotoxic activity with average IC50 values of 17.16 lM (van Dijk et al. 2011). Thereby, Cc-CATH3(1-29) may serve as an excellent candidate for therapeutic development given the better potency and breadth of antibacterial capacity. In different AMPs, the amino-terminal and carboxyterminal regions have been reported to play different role in terms of antimicrobial and cytotoxic activities. A certain part of amino acid residues of many AMPs can be omitted without interfering with their bacterial killing activity, and thus allowing a synthesis of shorter peptide sequences with retained activity at a lower production cost. For example, a number of researches have revealed that the antibacterial activity of human sole cathelicidin LL-37, which demonstrated potent and broad antimicrobial activity against a wide range of microorganisms (Pompilio et al. 2011; Kanthawong et al. 2012), was located in its mid-region and a truncation of up to 12 N-terminal residues of LL-37 had found to have no effect on its antibacterial activity (Li et al. 2006; Nell et al. 2006; Kanthawong et al. 2012). According to Li et al., residues 17-33 are the minimal antibacterial segment of LL-37 which remains active against Grampositive and Gram-negative bacteria. This shortest active fragment consists of an amphipathic a-helix with five cationic residues. Although hydrophobicity and amphipathicity are crucial for the antibacterial activity of AMPs, the cationic side chains on the hydrophilic surface were the most accountable (Li et al. 2006) in this case. This N-terminus segment with less proportion of cationic residues is
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dispensable for antibacterial activity of LL-37. This suggested that the electrostatic interactions of the cationic residues of LL-37 are with anionic phospholipids, which are ubiquitous in the bacterial cell membrane, are important for the antibacterial activity. In chicken cathelicidin, the C-terminus has found to play an essential role for antibacterial activity in fowlicidin-1 (CATH-1). An amino-terminal truncated analog of CATH1 of up to 4–7 residues retained its antibacterial property against different bacterial strains including methicillin-resistant S. aureus (MRSA), which was comparable to the full length peptide. The C-terminus helical segment of fowlicidin-1 (CATH-1) contain the bulk of cationic charges (?5) while an amphipathic a-helix contained a high proportion of positively charges (?8). Even though, Cc-CATH3(1-29) may serve as a novel antibiotic candidate, the long peptide with twenty nine residues may face the problem in production cost. Therefore, the objectives of this study are to investigate the necessary sequence promoting biological functions of the amino-terminal segments of Cc-CATH3(1-29) in terms of antibacterial activity, cytotoxicity, and membrane perturbation activity and to clarify how and to what extent the progressive deletion of four amino acids alter biological properties of the peptides and to possibly identify a shorter variant with retained or even improved therapeutic potential. This study also focused on the importance of N-terminus of Cc-CATH3(1-29) on its functions since there is no report about the important of N-terminal part of CcCATH3(1-29). This study will be beneficial for further design and development of Cc-CATH3 to be a novel antibiotic.
Materials and methods Chemicals Calcein disodium salt was obtained from Fluka, Switzerland. 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1-palmitoyl2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl2-oleoyl-sn-glycero-3-phospho-(10 -rac-glycerol) (sodium salt) (POPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) were purchased from Avanti polar lipids, USA. Sephadex G-25 Medium was bought from GE Healthcare, Sweden. Triton-X 100 was obtained from Panreac, Spain. Ampicillin sodium salt was purchased from Bio Basic, Canada. Mueller–Hinton Broth and agar powder were bought from HiMedia Laboratories, India. Dimethyl sulfoxide (DMSO) was bought from AMRESCO, USA. Dulbecco’s modified eagle medium (DMEM), penicillin–streptomycin,
Effect of N-terminal truncation on antibacterial activity, cytotoxicity and membrane…
and 0.25 % trypsin, and 0.1 % ethylenediaminetetraacetic acid (EDTA) were purchased from WISENT Inc, Canada. Fetal bovine serum (FBS) was bought from JR Scientific Inc, USA. Methylthiazol tetrazolium (MTT) and tryptan blue were purchased from Invitrogen, USA.
from the wells showing no visible sign of growth were inoculated onto sterile Mueller–Hinton (MH) Agar plates by streak plate method. The least concentration that showed no bacterial growth after an overnight incubation at 37 C was recorded as the minimum bactericidal concentration (MBC) value.
Peptide design and analysis Cell culture and cytotoxic activity A series of truncated variants of Cc-CATH3 was created by deleting four amino acids progressively from the amino terminal region. The amphipathicity of each peptide was demonstrated using helical wheel projections (performed with Heliquest) (Gautier et al. 2008). Cc-CATH3(1-29) and its three amino-terminal truncated analogues were commercially synthesized by China Peptides Co., Ltd (China) using standard solid-phase synthesis method to the purity of [98 % and characterized by mass spectrometry. All the lyophilized peptides were stored at -20 C. Deionized (DI) water was used as a solvent. Physicochemical properties and predicted helical structure of the peptides were also calculated using the Heliquest program (Gautier et al. 2008) and compute pI/MW tool program. Bacteria culture and antibacterial assay All the bacteria strains were purchased from the Culture Collection of the Department of Medical Sciences Thailand (DMST) under the Ministry of Public Health, Thailand. They were stored at -80 C until use. Subcultures of frozen bacteria were grown in Mueller–Hinton (MH) Broth overnight at 37 C in shaking incubator. The in vitro antibacterial activity of Cc-CATH3 and its N-terminally truncated variants were determined as the minimum inhibitory concentration (MIC) by broth microdilution assay as previously described (Otvos and Cudic 2007). In brief, an overnight bacteria culture was diluted with MHB to adjust turbidity, and the absorbance was measured spectrophotometrically at optical density 600 nm (T70 ? UV/ Vis spectrometer, PG Instruments Ltd.). The desired absorbance should be in the range of 0.08–0.13 (McFarland standard 0.5) which approximately represents a bacterial cell number of 1 9 108 CFUs/ml. The bacterial suspension was further diluted in MHB to 1 9 105 CFUs/ml. Then 180 lL aliquot of bacterial suspension was transferred to a flat-bottom 96-well microtiter plate per well with an addition of 20 lL of twofold serially diluted peptides to obtain final concentrations of 0.125, 0.25, 0.5, 1, 2, 4, and 8 lM in triplicate. Ampicillin and DI water were used as positive and negative controls respectively. After overnight incubation at 37 C, the MIC value of each peptide was determined by visual inspection; the lowest peptide concentration that gave no visible bacterial growth. To further determine bactericidal activity of each peptide, bacteria
The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was used to evaluate the cytotoxicity of Cc-CATH3 and its variants against cell line (Sieuwerts et al. 1995). The L929 mouse fibroblast cell line was cultured in a humidified atmosphere containing 5 % CO2 at 37 C in the respective media supplemented with 10 % FBS, 50 U/ml of penicillin/streptomycin. When reached 80 % confluence, the cell culture was trypsinized and plated in 96-well plates (1 9 105 cells/well). After 24 h incubation, 100 ll of twofold serial dilutions of each peptide solution were added in triplicate with maximum final concentration of 50 lM. 10 % DMSO in media and media were used as a positive and a negative control, respectively. Treated cells were further incubated for 24 h at 37 C in humidified 5 % CO2 atmosphere. After 24 h incubation, treatment medium was replaced with fresh medium containing 0.05 % w/v of MTT solution and further incubated for 4 h. The medium was then discarded, and 100 ll of DMSO was added to each well to solubilize formazan dye crystals produced by mitochondrial succinate dehydrogenase, which exists in living cells. The optical density of each sample was read on a microplate reader at 570 nm with a reference wavelength of 680 nm (T70 ? UV/Vis spectrometer, PG Instruments Ltd.). The percentage of viability was calculated as followed: % Cell viability ¼
Absorbance of sample 100 Absorbance of control
Preparation of large unilamellar vesicles (LUVs) and membrane leakage assay The membrane perturbation effects of each peptide will be investigated through LUVs calcein leakage assay (Zhang et al. 2010). The sixteen LUV models which are DOPC, DOPG, DOPC:DOPG (3:7), DOPC:DOPG (7:3), DOPE:DOPG (2:8), DOPE:DOPG (3:7), DOPE:DOPG (7:3), DOPE:DOPG (8:2), POPC:POPG (2:8), POPC:POPG (3:7) POPC:POPG (7:3), POPC:POPG (8:2), POPE:POPG (3:7), POPE:POPG (7:3) and POPE:POPG (2:8), and POPG were prepared by dissolving particular proportion of phospholipids in chloroform. The solvents are evaporated under a stream of nitrogen until a thin film is formed. The lipids films are subjected to a vacuum for 4 h and then thoroughly resuspended in 70 mM calcein solution in Hepes buffer
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(10 mM Hepes, 150 mM NaCl, 1 mM EDTA, pH 7.4). The liposome suspension is extruded through two polycarbonate filters with a 100-nm pore size for 50 times at room temperature. The unloaded calcein dye is separated from the liposomes by gel filtration using Sephadex G-25 medium column. The LUV dispersion was diluted with Hepes buffer to obtain phospholipids concentration of 25 lM in the well, and then an appropriate concentration of peptide solution was added to the mixture. Leakage of calcein from LUVs was tested in peptide-to-lipid ratio (P/L ratio) of 0.01. The calcein leakage assay was monitored after a 5-min incubation period by measuring fluorescence intensity at 520 nm (excitation at 490 nm). For determination of 100 % dye-release, 1 % of Triton X-100 solution in Hepes buffer was added to dissolve the vesicles. The percentage of dyerelease was determined using the following equation: F F0 % Leakage ¼ 100 Ft F 0 where F0 represents the background fluorescence intensity of the intact vesicles without addition of peptides, F is the fluorescence intensity measured after peptide addition, and Ft is fluorescence intensity of the lysed vesicle after addition of 1 % Triton X-100.
Results Peptide sequence analysis The sequences of Cc-CATH3 and its truncated variants are shown in Table 1. The amphipathicity of each peptide was illustrated by a helical wheel projection program using Heliquest (Gautier et al. 2008). The results demonstrated that the hydrophobic amino acids are concentrated on one side of a helix, whereas the charged and hydrophilic amino acids are present on the other side (Fig. 1). Physicochemical properties of the peptides molecular weight (MW), isoelectric point (pI), charge and hydrophobicity were also calculated using Compute pI/MW tool program and Heliquest program (Gautier et al. 2008) as shown in Table 1. The results illustrated that all truncated variants contain
similar values of pI and charge with the value of 11.10 and 4, respectively. Cc-CATH3(5-29) demonstrated the highest value in hydrophobicity. Antibacterial activity of Cc-CATH3 and its analogues The antibacterial results (Table 2) show that the full-length peptide Cc-CATH3(1-29) and Cc-CATH3(5-29) had the ability to inhibit growth of the investigated bacteria with relatively low concentrations. Cc-CATH3(1-29) displayed a strong antimicrobial activity against the tested microorganisms with the MIC values in the range of 2–4 lM. Interestingly, Cc-CATH3(5-29) outperformed the parent peptide with MICs between 1–2 lM. On the other hand, Cc-CATH3(9-29) and Cc-CATH3(13-29) exerted no antibacterial activities at the maximum tested concentration of 32 lM. Comparing between Cc-CATH3(1-29) and CcCATH3(5-29), the latter obviously demonstrated higher activity against all tested Gram-positive bacteria and was bactericidal at twofold MIC values for all tested Grampositive strains. Of the four peptides, Cc-CATH3(5-29) exhibited the strongest killing affect against most tested bacteria strains. These findings suggested that the deletion of four amino-terminal residues of the full length peptide resulted in an increase in antibacterial efficacy. However, the peptides seemed to have lost its antibacterial activity when they were further truncated by eight and twelve residues. Therefore a short motif of amino acid residues fifth to eighth ‘‘FWPL’’ is indispensable for the antibacterial activity Cytotoxic activity of the peptides Cytotoxicity activity of Cc-CATH3(1-29) and its three truncated analogs were assessed by MTT assay in L929 cell lines. The methylthiazol tetrazolium or MTT assay was conducted to determine cell viability or cytotoxicity had occurred to the cells following an exposure to the peptides. MTT is a water soluble salt that can penetrate into cells as well as mitochondria. The mitochondrial dehydrogenase of viable cells subsequently converts this compound into insoluble formazan crystals, the absorbance of which can be
Table 1 Amino acid sequences and physicochemical properties of Cc-CATH3 and its amino-terminal truncated variants Peptides
Sequences
Length (residue)
MW (dalton)
pI
Charge (z)
Hydrophobicity (H)
Cc-CATH3(1-29) Cc-CATH3(5-29)
RVRRFWPLVPVAINTVAAGINLYKAIRRK FWPLVPVAINTVAAGINLYKAIRRK
29 25
3379.11 2811.41
12.18 11.10
7 4
0.462 0.608
Cc-CATH3(9-29)
VPVAINTVAAGINLYKAIRRK
21
2267.75
11.10
4
0.416
Cc-CATH3(13-29)
INTVAAGINLYKAIRRK
17
1901.29
11.10
4
0.310
MW molecular weight, pI isoelectric point
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Effect of N-terminal truncation on antibacterial activity, cytotoxicity and membrane…
Fig. 2 A Cytotoxicity effect in L929 cells of Cc-CATH3(1-29) (filled diamond, solid line), Cc-CATH3(5-29) (filled square, dashed line), Cc-CATH3(9-29) (filled triangle, dotted line), and Cc-CATH3(13-29) (filled circle, dashed line). Results were reported as mean ± SD values
Fig. 1 Helical wheel projections of a Cc-CATH3(1-29), b CcCATH3(5-29), c Cc-CATH3(9-29), d Cc-CATH3(13-29). The arrow is a vector indicating the direction the hydrophobic moment, pointing towards the hydrophobic face of peptides
measured at 540 nm in a microplate reader. Cytotoxic activity of the peptides toward L929 cell lines was demonstrated in Fig. 2. Evidently, Cc-CATH3(1-29) and CcCATH3(5-29) displayed cytotoxicity in a dose response manner where the latter exerted a higher degree of toxicity. IC50 values were 9.33 and 4.93 lM, respectively. In contrast, Cc-CATH3(9-29) and Cc-CATH3(13-29) demonstrated low toxic to the cell with approximately no less than 80 % viability at all concentrations tested. Ability of Cc-CATH3 and its analogues to induce membrane leakage Degree of leakage as a function of peptide-to-lipid molar ratio The lowest concentration of peptides that can cause leakage was determined by titrating additional amount of peptides to LUVs composed of DOPC, DOPE:DOPG (3:7), Table 2 Antibacterial activity of Cc-CATH3 and its N-terminal truncated variants against Gram-positive bacteria
Peptides
and DOPE:DOPG (7:3). Each data point was recorded at 27 C after 5 min of incubation. The results illustrated that a high degree of calcein leakage from these three examined types of LUVs was induced by both Cc-CATH3(1-29) and Cc-CATH3(5-29) at a relatively low peptide-to-lipid (P/L) molar ratio. Apparently the degree of calcein release mediated by each peptide was distinguishable at 0.01 P/L ratio (Figs. 3, 4, 5). Figure 3 shows the level of induced calcein leakage from zwitterionic DOPC LUVs. Surprisingly, CcCATH3(1-29) induced calcein release from neutral DOPC, causing approximately 50 % leakage at 0.1 P/L ratio. The leakage caused by Cc-CATH3(5-29) was considerably higher, with approximately 80 % of dye release observed at only 0.025 P/L ratio and saturated at higher ratios. In contrast, Cc-CATH3(9-29) and Cc-CATH3(13-29) induced a very low leakage level from DOPC LUVs even up to a maximum tested P/L ratio of 0.1. Figures 4 and 5 illustrated the leakage extent of calcein entrapped in net negatively charged LUVs of DOPE:DOPG 3:7 and DOPE:DOPG 7:3 respectively. For Cc-CATH3(1-29), the data indicated that Cc-CATH3(1-29) induced higher leakage extents from both LUVs suspensions when compared to neutral DOPC vesicle. Moreover, the degree of leakage also appeared to be correlated with the ratio of anionic phospholipid DOPG. At 0.1 P/L ratio, Cc-CATH3(1-29)
Gram-positive bacteria S. Epidermidis DMST 15505
B. Cereus DMST 5040
MIC (lM)
MBC (lM)
MIC (lM)
MBC (lM)
Cc-CATH3(1-29)
4
4
2
4
Cc-CATH3(5-29)
2
2
1
2
Cc-CATH3(9-29)
[32
N/A
[32
N/A
Cc-CATH3(13-29)
[32
N/A
[32
N/A
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Fig. 3 The extent of calcein leakage from liposome composed of DOPC caused by caused by Cc-CATH3(1-29) (filled diamond, solid line), Cc-CATH3(5-29) (open square, dashed line), Cc-CATH3(9-29) (filled triangle, dotted line), and Cc-CATH3(13-29) (open circle, dashed line) as a function of peptide per lipid ratio. Results were reported as mean ± SE (n = 6)
caused approximately 80 and 60 % leakage in DOPE:DOPG 3:7 and DOPE:DOPG 7:3 respectively. For CcCATH3(5-29), leakage profiles were similar in manner to that in DOPC. At 0.025 P/L approximately as high as 80 % calcein release was induced and the leakage curves appeared to reach plateau at the higher ratios. Apparently, CcCATH3(5-29) caused a considerably higher extent of dye release than that of Cc-CATH3(1-29) at the same P/L molar ratio. Similar to the result in DOPC, Cc-CATH3(929) and Cc-CATH3(13-29) still induced a very low leakage level from these two types of net negatively charged LUVs. Up to a maximum tested P/L molar ratio of 0.1, which is extremely high from a physiological aspect, Cc-CATH3(929) and Cc-CATH3(13-29) induced less than 15 % leakage (Figs. 3, 4 and 5). Effect of phospholipid head groups on calcein leakage from 1,2-dioleoyl (DO) acyl chain liposomes
Fig. 4 The extent of calcein leakage from liposome composed of DOPE:DOPG 3:7 caused by Cc-CATH3(1-29) (filled diamond, solid line), Cc-CATH3(5-29) (open square, dashed line), Cc-CATH3(9-29) (filled triangle, dotted line), and Cc-CATH3(13-29) (open circle, dashed line) as a function of peptide per lipid ratio. Results were reported as mean ± SE (n = 6)
Fig. 5 The extent of calcein leakage from liposome composed of DOPE:DOPG 7:3 caused by Cc-CATH3(1-29) (filled diamond, solid line), Cc-CATH3(5-29) (open square, dashed line), Cc-CATH3(9-29) (filled triangle, dotted line), and Cc-CATH3(13-29) (open circle, dashed line) as a function of peptide per lipid ratio. Results were reported as mean ± SE (n = 6)
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The effect of phospholipid head groups of 1,2-dioleoyl (DO) acyl chain liposomes on calcein leakage assay inducing by the peptides were examined. The assays were performed in liposomes mixed between phospholipids containing different polar head groups containing the same 1,2-dioleoyl (DO) acyl chains, (18:1n-9)(18:1n-9), namely; DOPC, DOPE, and DOPG. As shown in Fig. 6, calcein fluorescence signal rapidly increased after an addition of Cc-CATH3(1-29) to neutral DOPC vesicles and the leakage curve reached a plateau of only 50 % leakage. However, when 30 % DOPC was replaced by negatively charged DOPG, the amount of leakage was significantly reduced and further decreased in the mixed lipids with 70 % DOPG. It was obvious that increasing the ratio of DOPG further decreased the leakage efficiency. Similarly, the effect of Cc-CATH3(1-29) on anionic DOPG vesicles was quite weak inducing only about 10 % calcein release. Likewise, Cc-CATH3(5-29) caused a similar pattern of the leakage curves; however, with an extremely higher degree of membrane disruption and faster kinetics than that of Cc-CATH3(1-29). In contrast, CcCATH3(9-29) and Cc-CATH3(13-29) induced a very low extent of leakage from the vesicles even after 25 min regardless of a ratio of DOPC or DOPG (data not shown). These results suggested that both Cc-CATH3(1-29) and CcCATH3(5-29) were membrane active, inducing membrane rupture with fast kinetics where the latter was apparently more efficient. Interestingly, they were not selective to negatively charged phospholipids. In net negatively charged LUVs composed of DOPE:DOPG, Cc-CATH3(9-29) and Cc-CATH3(13-29) caused low level of membrane leakage similar to the leakage results in DOPC:DOPG (data not shown). Figure 7 showed that a high degree of calcein leakage was caused by both Cc-CATH3(1-29) and Cc-CATH3(5-29) where the higher
Effect of N-terminal truncation on antibacterial activity, cytotoxicity and membrane…
Fig. 6 The extent of calcein leakage caused by a Cc-CATH3(1-29) and b Cc-CATH3(5-29) from liposomes composed of DOPC (filled diamond, line), DOPC:DOPG 7:3 (open square, dashed line), DOPC:DOPG 3:7 (filled triangle, dotted line), DOPG (open circle, line) as a function of time. The peptide-to-lipid ratio was 0.01 and the final lipid concentration was 25 lM. Results were reported as mean ± SE (n = 6)
the ratio of zwitterionic DOPE had the higher the leakage extent. However, Cc-CATH3(5-29) induced roughly faster and a bigger disruption. These results confirmed that CcCATH3(5-29) was more efficient than Cc-CATH3(1-29) in disrupting membrane vesicles. Nevertheless, the lytic activity of both peptides did not depend on the ratio of negatively charged DOPG. Effect of phospholipid head groups on calcein leakage from 1-palmitoyl 2-oleoyl (PO) acyl chains liposomes The amount of calcein release was also determined in mixed phospholipids containing 1-palmitoyl 2-oleoyl (PO) acyl chains, (16:0)(18:1n-9), with different polar head groups. Similar to the leakage results in the DO lipids, all types of the examined PO lipids were barely leaked by CcCATH3(9-29) and Cc-CATH3(13-29) (data not shown). Conversely, both Cc-CATH3(1-29) and Cc-CATH3(5-29) induced a high degree of leakage in general. In mixed PO liposomes composed of one type of anionic lipid e.g. POPC:POPG and POPE:POPG, the pattern of the leakage curves were similar to those of the DO series in that the less
Fig. 7 The extent of calcein leakage caused by a Cc-CATH3(1-29) and b Cc-CATH3(5-29) from liposomes composed of DOPE:DOPG 8:2 (filled diamond, line), DOPE:DOPG 7:3 (open square, dashed line), DOPE:DOPG 3:7 (filled triangle, dotted line), DOPE:DOPG 2:8 (open circle, line) as a function of time. The peptide-to-lipid ratio was 0.01 and the final lipid concentration was 25 lM. Results were reported as mean ± SE (n = 6)
the ratio of anionic PG the higher the leakage extent. CcCATH3(5-29) yet again demonstrated slightly higher lytic activity than Cc-CATH3(1-29) (see Figs. 8, 9). Effect of acyl-chain composition on calcein leakage from 1-palmitoyl 2-oleoyl (PO) acyl chains liposomes To better understand the influence of phospholipid acylchain composition on the membrane leakage induction of Cc-CATH3(1-29) and its truncated variants, we compared leakage profiles of the peptides in liposomes containing different type of acyl chain but the same head group which are DOPG and POPG. As shown in Fig. 10, the release of calcein from DOPG and POPG liposomes induced by CcCATH3(1-29) and Cc-CATH3(5-29) followed a similar pattern, however; the leakage extent and kinetics depended on phospholipid acyl-chain of the vesicles. The modification of acyl-chain structure from DO to PO alters the ability of both peptides to cause greater and faster membrane disruption.
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Fig. 8 The extent of calcein leakage caused by a Cc-CATH3(1-29) and b Cc-CATH3(5-29) from liposomes composed of POPC:POPG 8:2 (filled diamond, line), POPC:POPG 7:3 (open square, dashed line), POPC:POPG 3:7 (filled triangle, dotted line), POPC:POPG 2:8 (filled circle, line), and POPG (x, dotted line) as a function of time. The peptide-to-lipid ratio was 0.01 and the final lipid concentration was 25 lM. Results were reported as mean ± SE (n = 6)
Fig. 9 The extent of calcein leakage caused by a Cc-CATH3(1-29) and b Cc-CATH3(5-29) from liposomes composed of POPE:POPG 8:2 (filled diamond, line), POPE:POPG 7:3 (open square, dashed line), POPE:POPG 3:7 (filled triangle, dotted line), and POPE:POPG 2:8 (open circle, line), as a function of time. The peptide-to-lipid ratio was 0.01 and the final lipid concentration was 25 lM. Results were reported as mean ± SE (n = 6)
Discussion
antibacterial potency. However, further truncation of CcCATH3(1-29) by eight and twelve residues strongly reduced the antibacterial efficacy. These data are similar to our previous study (Ngamsaithong et al. 2012). In previous study, the peptides were tested in Gram-positive and Gramnegative bacteria which are S. aureus, B. subtilis, E. coli and S. typhimurium. The results indicated that CcCATH3(5-29) contain the highest antibacterial activity. However, Cc-CATH3(9-29) and Cc-CATH3(13-29) illustrated no antibacterial activity. From Table 1, although all 3 truncated analogues have net ?4 charge, which decrease from ?7 of Cc-CATH3(1-29), only Cc-CATH3(5-29) maintains its antibacterial activity and even displays greater potency than the full length peptide. It is obvious that the first four residues at the N-terminus ‘‘RVRR’’, contributing ?3 to the total peptide charge, is not essential with respect to its antimicrobial property. Therefore, the absence of this short segment in Cc-CATH3(5-29) causes no effect to its bacteria killing ability. In contrast, residues
Interaction of AMPs with biological membranes involves several steps including initial recognition and binding of peptide and membrane conformational changes of the peptide, insertion into the membrane to generate pores and membrane rupture, leading to cell death (Yeaman and Yount 2003; Brogden 2005; Shai 1999). The initial recognition is mainly by ionic interaction between the charged group of phospholipid and charged group of peptide, while the insertion of the antimicrobial peptide processes is by the hydrophobic interaction between the hydrophobic tail of phospholipids and the nonpolar amino acid composing the peptide. In this study, antibacterial assay confirms that Cc-CATH3(1-29) is potent against Gram-positive bacteria, similar to the previously reported studies (Feng et al. 2011; Ngamsaithong et al. 2012). It also reveals that the absence of the first four amino-terminal residues of the parent peptide results in an improved
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Effect of N-terminal truncation on antibacterial activity, cytotoxicity and membrane…
Fig. 10 The extent of calcein leakage caused by a Cc-CATH3(1-29) and b Cc-CATH3(5-29) from DOPG (filled diamond, dashed line) and POPG (open square, solid line) liposomes as a function of time. The peptide-to-lipid ratio was 0.01 and the final lipid concentration was 25 lM. Results were reported as mean ± SE (n = 6)
five to eight of ‘‘FWPL’’ are indispensable as the exclusion of this short motif in Cc-CATH3(9-29) and Cc-CATH3(1329) leads to a complete loss of antibacterial activity at the maximum tested concentration of the peptides. So it is possible to conceive that higher antibacterial potency of CcCATH3(5-29) contributes to the explanations that the remaining ?4 amino residues at the C-terminus may be sufficient to cause electrostatic interaction with a net negative charge on the bacterial membrane surface in an initial step of peptide/membrane interaction. This ‘‘FWPL’’ fragment that contains strong hydrophobic residues of Phe and Trp allows the peptide to hydrophobically interact with bacterial membrane in an insertion step to form pore and rupture the membrane (Haney et al. 2012). Furthermore, the shorter sequence of Cc-CATH3(5-29) composed of 25 residues may provide an optimal length for the peptide to remain stably inserted in the hydrophobic core of the bacterial membrane during the process of bacterial intracellular efflux. For cytotoxicity, it is clear that cytotoxic activity of the four peptides is in line with antibacterial activity. In general, cytotoxic activity of Cc-CATH3(1-29) and its truncated analogues parallels their antibacterial effects, but
requires higher concentrations. Cc-CATH3(1-29) is rather toxic but the exclusion of four N-terminal residues ‘‘RVRR’’ from this parent peptide brings about even stronger toxicity as exhibited by Cc-CATH3(5-29), suggesting that this ‘‘RVRR’’ short sequence has no effect on the cytotoxicity. In contrast, the deletion of eight and twelve residues from N-terminus results in the loss of cytotoxic efficacy. Therefore the fragment of ‘‘FWPL’’, which plays vital role in antibacterial activity, is critical in governing cytotoxic activity as well. Explanations behind this result are likely to be similar to that of the antimicrobial assay, irrespective to prokaryotic or eukaryotic membranes. Following an initial step of electrostatic attraction between peptide and membranes, the presence of strong hydrophobic amino acids of Phe and Trp at flanking position of Cc-CATH3(5-29) then drive the peptide insertion into the interfacial region of the bilayer membrane and into the hydrophobic core while its length of 25 residues is possibly optimal for the peptide to remain stably attached to the membrane. For membrane destabilization analysis, the experiments were performed in membrane models which may not be perfectly representative models for bacterial membranes. However, the membrane destabilization analysis is a method that can investigate and predict the ability in membrane perturbation of AMPs. In this analysis, it appears that membrane disruption ability of Cc-CATH3(129) does not depend on negatively charged head groups of lipids as anticipated but seems to correlate with zwitterionic lipids instead. As can be seen from the leakage profiles (Figs. 6, 7), the effect of the peptide on anionic DOPG vesicles is considerably weak and becomes stronger when the ratio of neutral lipids of DOPC and DOPE are increased in mixed DOPC:DOPG and DOPE:DOPG, respectively. This observation is in contrast with the case of human cathelicidin LL-37 which is reported to be selective to anionic lipids and unable to induce leakage of neutral DOPC vesicles (Zhang et al. 2010). Thus, it is possible that electrostatic interaction might not play such a determining role in the lytic activity of Cc-CATH3(1-29). When focusing on the effect of the composition of neutral phospholipids, Cc-CATH3(1-29) efficiency in inducing calcein leakage is generally increased when the phospholipid headgroup composition is varied from DOPC to DOPE at the same molar ratio (Figs. 6, 7). Since it is accepted that most AMPs act by destabilizing membranes and a major step to disintegrate membranes involves the ability of peptides to insert into the membrane and induce pore formation. Basically a trans-membrane pore has a negative Gaussian curvature while the insertion of AMP into the lipid membrane causes positive curvature along the membrane surface (Wei et al. 2011; Huang 2006). Therefore, this leakage increase may arise from the existence of DOPE lipid which could help achieve negative curvature
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along the rim of a pore, leading to the generation of negative Gaussian curvature necessary for trans-membrane pore formation (Nishizawa and Nishizawa 2010). Therefore, the result suggests that the intrinsic membrane curvature, rather than membrane charge, may play a major role in the membrane-disrupting activity of Cc-CATH3(129). However, when taking the antibacterial and cytotoxic assay results into consideration, this could not explain why Cc-CATH3(1-29) is non-preferential over prokaryotic membranes which contain higher concentrations of negative-curvature lipids such as PE than do eukaryotic membranes. Nonetheless, our result is in contrast with the leakage study of ANTS/DPX-entrapped liposomes by GALA peptide since changing the phospholipids headgroup from cylindrical-shaped PC to inverted-cone shaped PE leads to the formation of vesicles with larger curvature which is likely to be more difficult to disrupt (Nicol et al. 2000). Cc-CATH3(1-29) efficiency in inducing calcein leakage from LUVs is influenced by the structure of the acyl chains (Fig. 10). At the same P/L ratio, Cc-CATH3(129) is less efficient at inducing leakage from liposomes composed of 1,2-dioleoyl-phosphatidylglycerol (DOPG) (18:1n-9)(18:1n-9) than from vesicles made of 1-palmitoyl 2-oleoyl-phosphatidylglycerol (POPG) (16:0)(18:1n-9). However, since the cis unsaturated acyl chains of DO lipids are more fluid than the PO acyl chains, this phenomenon cannot be explained solely by modifications of order of the lipid acyl chains. The leakage results of Cc-CATH3(5-29) share a similar trend with that in Cc-CATH3(1-29); exhibits no preferential binding to anionic lipids, causes greater dye release to DOPE than DOPC (Figs. 6, 7) as well as in PO lipids than DO lipids (Fig. 10). Despite that, the overall membrane rupturing ability of Cc-CATH3(5-29) becomes fairly stronger, which is in agreement with their greater antibacterial and cytotoxic activities. These leakage assays further suggest that the four amino-terminal residues are expendable. Since dye release caused by both CcCATH3(1-29) and Cc-CATH3(5-29) are observed at low peptide per lipid ratios, the peptides being relatively long, it is unlikely that the peptides disintegrate membranes via the carpet mechanism. Subsequent to the deletion of four dispensable amino acids ‘‘RVRR’’ from N-terminal that allows strong hydrophobic Phe and Trp in ‘‘FWPL’’ to be located at flanking position, lytic efficiency of CcCATH3(5-29) is improved. Being 25-residues in length, Cc-CATH3(5-29) may be optimal for the peptide to remain stably inserted in the membrane bilayer. For these reasons, a pore formation mechanism is the most likely mechanism for both Cc-CATH3(1-29) and Cc-CATH3(5-29) at least in our tested model membranes. In conclusion, we have highlighted that ‘‘FWPL’’ are important for activities and the residues F and W may be
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major responsible for this phenomenon, the further studies such as examining the activities of mutant peptides in this rigion should be performed in order to better understand in structure–function relationship of this peptide. From our results, it is likely that Cc-CATH3(1-29) acts via pore formation mechanism, though further tests should be examined. Acknowledgments This project is supported by the Office of the High Education Commission and Mahidol University under the National Research Universities Initiative. Conflict of interest of interest.
The authors declare that there are no conflicts
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