- Email: [email protected]
A novel antimicrobial peptide derived from membrane-proximal external region of human immunodeficiency virus type 1
Accepted Manuscript A novel antimicrobial peptide derived from membrane-proximal external region of human immunodeficiency virus type 1 Xiaoqiu He, Huayan Zhang, Yuhua Shi, Xin Gong, Shanshan Guan, He Yin, Lan Yang, Yongjiao Yu, Ziyu Kuai, Dongni Liu, Rui Hua, Song Wang, Yaming Shan PII:
S0300-9084(16)00049-3
DOI:
10.1016/j.biochi.2016.02.006
Reference:
BIOCHI 4938
To appear in:
Biochimie
Received Date: 15 December 2015 Accepted Date: 9 February 2016
Please cite this article as: X. He, H. Zhang, Y. Shi, X. Gong, S. Guan, H. Yin, L. Yang, Y. Yu, Z. Kuai, D. Liu, R. Hua, S. Wang, Y. Shan, A novel antimicrobial peptide derived from membrane-proximal external region of human immunodeficiency virus type 1, Biochimie (2016), doi: 10.1016/j.biochi.2016.02.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
ABSTRACT With increasing microbial drug resistance worldwide, antimicrobial peptides (AMPs) are considered promising alternatives to addressing this problem. In this study, a series of synthetic
RI PT
peptides were designed based on the membrane-disrupting properties of the membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) envelope protein. The peptide AP16-A was found to exhibit the most effective antimicrobial activities against both
SC
Gram-negative and Gram-positive bacteria. The minimal bactericidal concentration (MBC) of AP16-A ranged from 2 µg/ml to 16 µg/ml. AP16-A had no detectable cytotoxicity in various tissue cultures and a mouse model. Furthermore, results of confocal fluorescence microscopy and
M AN U
the SYTOX Green uptake assay indicated that AP16-A killed Gram-negative bacteria by the combined effects of relatively slow membrane permeabilization and interaction with an intracellular target, while it killed Gram-positive bacteria by a fast membrane permeabilization process, which achieved relatively more rapid bacterial killing kinetics. The results of this study support the potential use of AP16-A as an AMP.
AC C
EP
killing kinetics
TE D
Keywords: antimicrobial peptides, antimicrobial activity, membrane permeabilization, bacterial
1
ACCEPTED MANUSCRIPT
A novel antimicrobial peptide derived from membrane-proximal
RI PT
external region of human immunodeficiency virus type 1
Xiaoqiu Hea, Huayan Zhanga, Yuhua Shia, Xin Gonga, Shanshan Guana, He Yina, Lan Yanga, Yongjiao Yua, Ziyu Kuaia, Dongni Liua, Rui Huab, Song Wangc, and Yaming Shana,d*
National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University,
Changchun, Jilin, China;
b
SC
a
Department of Hepatology, The First Hospital, Jilin University,
Changchun, Jilin, China; c State Key Laboratory of Theoretical and Computational Chemistry,
M AN U
Institute of Theoretical Chemistry, Jilin University, Changchun, Jilin, China; d Key Laboratory for Molecular Enzymology and Engineering, The Ministry of Education, School of Life Sciences, Jilin University, Changchun, Jilin, China
EP
TE D
*Address correspondence to Yaming Shan, [email protected]
AC C
Abbreviations: AMPs: antimicrobial peptides, MPER: membrane-proximal external region, HIV-1: human immunodeficiency virus type 1, LPS: lipopolysaccharides, CFU: colony-forming units, MBC: minimal bactericidal concentration, OM: outer membrane
1
ACCEPTED MANUSCRIPT
ABSTRACT With increasing microbial drug resistance worldwide, antimicrobial peptides (AMPs) are considered promising alternatives to addressing this problem. In this study, a series of synthetic
RI PT
peptides were designed based on the membrane-disrupting properties of the membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) envelope protein. The peptide AP16-A was found to exhibit the most effective antimicrobial activities against both
SC
Gram-negative and Gram-positive bacteria. The minimal bactericidal concentration (MBC) of AP16-A ranged from 2 µg/ml to 16 µg/ml. AP16-A had no detectable cytotoxicity in various tissue cultures and a mouse model. Furthermore, results of confocal fluorescence microscopy and
M AN U
the SYTOX Green uptake assay indicated that AP16-A killed Gram-negative bacteria by the combined effects of relatively slow membrane permeabilization and interaction with an intracellular target, while it killed Gram-positive bacteria by a fast membrane permeabilization process, which achieved relatively more rapid bacterial killing kinetics. The results of this study support the potential use of AP16-A as an AMP.
AC C
EP
killing kinetics
TE D
Keywords: antimicrobial peptides, antimicrobial activity, membrane permeabilization, bacterial
2
ACCEPTED MANUSCRIPT
1.
Introduction The accelerated emergence of drug-resistant bacterial strains caused by the extensive use of
traditional antibiotics is a current major threat [1, 2]. Therefore, the development of a more
RI PT
effective treatment to overcome the problem of drug resistance is urgently needed. Antimicrobial peptides (AMPs) can provide a possible alternative to traditional drugs with a new mode of action and remarkable therapeutic effects [3, 4]. Generally, naturally occurring AMPs contain 12–50
SC
amino acids in length, with an overall positive charge and an amphipathic structure. Most AMPs can directly bind to the bacterial membrane and kill the organism by accessing intracellular targets through membrane permeabilization [5-8]. The net positive charge facilitates the
M AN U
interaction between AMPs and negatively charged microbial surfaces, with the amphipathic secondary structure allowing AMPs to incorporate into microbial membranes [9]. To date, more than 1,000 AMPs have been identified in various species, including plants, insects, fish, frogs and mammals [10]. However, most natural AMPs are large, have low potency and are toxic to host cells [11]. Considering these complications, different synthetic AMPs, as well as corresponding
TE D
mimics, have been the focus of considerable interest in studies aimed at increasing the effectiveness of AMPs [12, 13]. Short designer AMPs that are less likely to induce resistance and
[4].
EP
show low toxicity to host cells and tissues have the potential to be the most effective candidates
The essential process of HIV-1 entry into a cell is the fusion of the viral and target cell
AC C
membranes. This crucial step is mediated by the HIV-1 envelope protein [14]. The membrane-proximal external region (MPER, Ac-665WASLWNWFNITNW LWYIK683-NH2), a highly conserved domain of the HIV-1 gp41 ectodomain, plays a critical role in viral envelope glycoprotein mediated-fusion and infectivity [15, 16]. In addition, the epitopes of well-characterized neutralizing antibodies 2F5, Z13 and 4E10 overlap this conserved region. Previous reports have shown that the MPER, with an unusually high percentage of tryptophan (Trp) residues, likely contributes to the membrane-disrupting properties [15]. Indolicidin, an 3
ACCEPTED MANUSCRIPT
analog of MPER, is also capable of disrupting membranes and has an unusually high number of Trps as well. Indolicidin is 13 amino acids (ILPWKWPWWPWRR) in length and is a naturally occurring AMP isolated from bovine neutrophils [17]. However, a high Trp content in AMPs is
RI PT
associated with high hemolysis, making indolicidin unsuitable for use as an antimicrobial agent [18]. Based on the membrane-disrupting properties of the MPER, we hypothesized that synthetic peptides derived from this region, such as the 4E10 epitope (Ac-NWFNITNWLWYIK-NH2), may
SC
have functional properties similar to those of known AMPs.
Although the sequences of AMPs vary greatly, certain amino acids, such as lysine (Lys) or arginine (Arg), are key components of AMPs [19]. In particular, Lys-rich AMPs exhibit rapid,
M AN U
nonhemolytic, broad-spectrum microbicidal properties [20, 21]. Thus, we aimed to increase this activity by adding Lys to the C-terminus of the 4E10 epitope to generate an AMP with 16 amino acids (AP16: Ac-NWFNITNWLWYIKKKK-NH2). In this study, a series of mutant peptides of AP16 were synthesized, and their antimicrobial activities in vitro against Gram-negative and Gram-positive bacteria were determined. Among them, AP16-A exhibited the highest
TE D
antimicrobial activity. Potential mechanisms for bactericidal activity were also investigated. By showing low toxicity in eukaryocytes and hemolytic activity, AP16-A has the potential for further development and use as an antibiotic. Materials and methods
EP
2.
2.1 Bacteria and reagents
AC C
Escherichia coli DH5α was purchased from Takara. Bacillus subtilis ATCC 6633 and Staphylococcus epidermidis ATCC 12228 were purchased from the American Type CultureCollection
(ATCC).
Melittin,
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium
bromide (MTT) and lipopolysaccharides (LPS) were purchased from Sigma-Aldrich. Poly-L-lysine and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Roster. SYTOX Green were purchased from KeyGEN BioTECH. N-phenyl-1-napthylamine (NPN) was purchased from Aladdin. 4
ACCEPTED MANUSCRIPT
2.2 Animals and ethics statement
Four weeks old BALB/c female mice(18−20 g), were purchased from the Changchun Institute of Biological Products Co., Ltd. The animal trials in this study were carried out
RI PT
in accordance with the Regulations and the Administration of Affairs Concerning
Experimental Animals approved by the State Council of People’s Republic of China (11-14-1988). The Institutional Animal Care and Use Committee (IACUC) of Jilin
SC
University approved all animal procedures (permit number: SCXK 2013-0001). 2.3 Peptides
All peptides used in this study (listed in Table 1) were synthesized by the Chinese Peptide
M AN U
Company (Hangzhou, China). Vendors provided data on peptide characterizations, including high-performance liquid chromatography (>98% purity) and mass spectrometry. All lyophilized peptides were dissolved in water to 1 mg/ml stocks and stored at −20°C. 2.4 Antimicrobial activity
Antimicrobial activity was determined using a method similar to that described previously
TE D
[22-24]. Bacterial suspensions were cultured to mid-logarithmic phase at 37°C and washed by several cycles of centrifugation and resuspension in 10 mM potassium phosphate buffer (PB), pH 7.2. Bacteria were diluted to between 1 × 106 colony-forming units (CFU)/ml and 2 × 106 CFU/ml
EP
in PB and then treated in the assay. Peptides were diluted two-fold in 96-well plates in the same buffer. One hundred microliters of bacteria was mixed with 100 µl of peptides, followed by
AC C
incubation at 37°C for 2 h without shaking. At the end of the incubation period, a 100 µl aliquot from each dilution was plated on tryptic soy agar and allowed to grow at 37°C overnight for the measurement of the minimal bactericidal concentration (MBC). The MBC is defined as the lowest peptide concentration on the agar plate, which exhibited no bacterial growth (zero colony). All peptides were tested in triplicate. 2.5 Dose-dependent bacterial killing 5
ACCEPTED MANUSCRIPT
Bacteria and peptides were prepared as in the antimicrobial activity assay. Serial 10-fold dilutions of control and test wells were included. A 100 µl aliquot from each dilution was plated on tryptic soy agar and incubated overnight at 37°C. Colonies of surviving bacteria were counted
RI PT
to determine the extent of peptide-induced killing under each concentration of peptides. 2.6 Hemolytic assay
The hemolytic activities of peptides were determined by hemolysis of guinea pig red blood
SC
cells. Freshly collected heparinized guinea pig blood was centrifuged at 1000 × g for 30 min. The erythrocytes were washed two times with saline and centrifuged again at 1000 × g for 10 min. A solution of 4% (v/v) guinea pig red blood cells was prepared and mixed with serial dilutions of
M AN U
peptides in saline in 96-well plates. The mixture was then incubated for 1 h at 37°C. After centrifugation at 1000 × g for 30 min, the percentage of hemolysis was determined by measuring the absorbance of the supernatant at a wavelength of 450 nm. Cells incubated with saline alone served as the negative control, and guinea pig red blood cell sample lysed with 1% Triton X-100
2.7 In vitro cytotoxicity
TE D
was used to represent 100% lysis (positive control).
The cytotoxicity of the peptides to hCMEC/D3 cells was measured by the MTT assay. Briefly, cells were seeded at 104 cells/well in 96-well plates at 37°C overnight. The next day, serial
EP
dilutions of peptides were added to each well. The untreated cells served as the negative control, and melittin, the major toxin of bee venom, was used as a positive control [25]. After 3 h of
AC C
incubation, 10% MTT solution (5 mg/ml) in phosphate-buffered saline was added, and the plate was incubated for 2 h with 5% CO2 at 37°C. The supernatant was then removed, and 150 µl dimethyl sulfoxide was added. The absorbance of the supernatant at a wavelength of 490 nm was measured.
2.8 In vivo cytotoxicity
BALB/c female mice were injected intraperitoneally (i.p.) with AP16-A (10 and 20 mg/kg of body weight) in saline to test the acute toxicity of this peptide in vivo. Peptides 6
ACCEPTED MANUSCRIPT
were administered in 200 µl saline, and the control were injected i.p. with an equal volume of saline. Each group consisted of six mice. After peptide injection, the number of dead mice and the change in body weight were recorded daily for 7 days
RI PT
post-injection. 2.9 Killing kinetics
Bacteria (1 × 106 CFU/ml to 2 × 106 CFU/ml) were treated with peptides at the MBC in PB. At
SC
the indicated time, aliquots of this mixture were serially diluted with Luria broth, which substantially inhibited the killing process, and then placed on tryptic soy agar plates and
of peptide exposure time. 2.10 Confocal fluorescence microscopy
M AN U
incubated overnight for viability measurement. The number of colonies was plotted as a function
The cellular localization of AP16-A was observed by confocal fluorescence microscopy. Bacteria were grown to the mid-logarithmic phase and then harvested, washed and resuspended in PB to a concentration of approximately 108 CFU/ml. The suspension was mixed with fluorescein
TE D
isothiocyanate (FITC)-labeled AP16-A solution to yield a final peptide concentration corresponding to 0.5 × MBC. Following incubation at 37°C, the cells were harvested by centrifugation, washed with PB and smeared on a poly-L-lysine-coated slide. Localization of
EP
labeled peptide was monitored under an inverted confocal microscope equipped with a 63× oil immersion lens (CarlZeiss LSM 710).
AC C
2.11 SYTOX Green uptake assay
Briefly, bacterial suspensions of 107 CFU/ml were prepared and mixed with 1 µM SYTOX Green for 15 min in the dark. After the addition of AP16-A to the final concentration corresponding to the MBC, uptake of SYTOX Green was determined using a fluorescence spectrophotometer (filter wavelengths of 485 and 520 nm for excitation and emission, respectively). 2.12 Gel retardation assay 7
ACCEPTED MANUSCRIPT
Briefly, AP16-A was incubated with pET-20b plasmid DNA at different N/P ratios (0, 0.2, 0.4, 0.6, 0.8, 1, 2 and 3) for 30 min at 37°C to form peptide/DNA complexes. The N/P ratio was defined as the proportion of amino nitrogen (NH3+) of AP16-A relative to the phosphate (PO4−) of
2.13 Outer membrane (OM) permeabilization assay
RI PT
DNA [26]. These complexes were analyzed by electrophoresis on a 1% agarose gel in TAE.
The membrane-permeabilizing activity of the peptide was determined by using the fluorescent
SC
dye NPN assay, as described previously [27]. Bacterial suspensions of 107 CFU/ml were prepared and mixed with 10 µM NPN, and uptake of the dye was determined using a fluorescence spectrophotometer (filter wavelengths of 350 and 420 nm for excitation and emission,
M AN U
respectively). The increase in fluorescence due to partitioning of NPN uptake into the OM was determined by increasing the concentration of the peptide. All experiments were conducted three times.
2.14 Assay of in vitro binding between AP16-A and LPS
Bacteria were prepared as for the antimicrobial activity assay. LPS from E. coli was
TE D
mixed with E. coli and AP16-A (1× MBC) for 2 hours. Serial 10-fold dilutions of control and test wells were included. A 100 µl aliquot from each dilution was plated on tryptic soy agar and incubated overnight at 37°C. Colonies of surviving bacteria were counted to
3.
Results
EP
determine the peptide-induced killing after adding LPS.
AC C
3.1. Antimicrobial activity
The antimicrobial activities of AP16 and its analogs against E. coli, B. subtilis, and S. epidermidis are shown in Table 2. AP16-A showed high broad-spectrum antimicrobial activity against both Gram-negative and Gram-positive bacteria. The MBCs of AP16-A were lower than 4 µg/ml for B. subtilis and 8 µg/ml for S. epidermidis. E. coli was the least sensitive to AP16-A (MBC = 16 µg/ml). As indicated in Fig. 1, bacterial killing by AP16-A was dose-dependent, further validating its antimicrobial activity. AP16-A exhibited more than a 2 log reduction in 8
ACCEPTED MANUSCRIPT
colony counts (>99% of bacteria killed) for E. coli and S. epidermidis at one half of the MBC. For B. subtilis, AP16-A exhibited more than a 4 log reduction in colony counts (>99.99% of bacteria killed) at one half of the MBC and 2 log reduction in colony counts (>99% of bacteria killed) at
RI PT
one fourth of the MBC. 3.2. In vitro and in vivo cytotoxicities
Several AMPs exhibited cytolytic activities. As shown in Fig. 2A, no evident hemolysis by
SC
AP16-A was observed at a final peptide concentration corresponding to 1 × MBC for all bacteria. However, melittin was approximately 72% hemolytic for the MBC of S. epidermidis and approximately 100% hemolytic for the MBC of E. coli and B. subtilis. The standard MTT assay
M AN U
was conducted to determine the cytotoxicity of AMPs to human cells (hCMEC/D3). As shown in Fig. 2B, the concentration of peptides ranged from 1 µg/ml to 128 µg/ml. The cells treated with melittin were completely killed at a low dose (8 µg/ml). By contrast, AP16-A caused only a low level of cytotoxicity at the concentration of 128 µg/ml. Female mice were injected i.p. with AP16-A (10 and 20 mg/kg of body weight) to determine the cytotoxicity of AP16-A in the animal
TE D
model. All mice were alive after seven days (Fig. 2C), and the weight of all mice were apparently unchanged compared with the control (Fig. 2D). 3.3. Kinetics of antimicrobial activity
EP
The tested bacteria were treated with AP16-A at concentrations corresponding to the MBC to determine the time course of bacterial killing (Table 1). Results showed that killing of B. subtilis
AC C
and S. epidermidis was more rapid than E. coli. B. subtilis was immediately killed within 5 min, and S. epidermidis was killed within 20 min upon the addition of AP16-A. However, complete killing of E. coli, a Gram-negative bacteria, by AP16-A occurred over the course of 100 min (Fig. 3).
3.4. Bactericidal mechanisms of AP16-A The localization of peptides in bacteria was examined by using FITC-labeled analogs corresponding to 0.5 × MBC to gain insight into the bactericidal mechanisms of AP16-A. Upon 9
ACCEPTED MANUSCRIPT
peptide treatment, the green fluorescence (FITC-labeled AP16-A) surrounded the blue fluorescence (nucleic acids), indicating that AP16-A accumulated on the membrane (Fig. 4A). The SYTOX Green uptake assay was conducted to further examine the effect of AP16-A on
RI PT
membranes. SYTOX Green is a membrane-impermeable nucleic acid stain that enters only membrane-disrupted cells. The results showed that a significant degree of membrane permeabilization was induced by the peptide on E. coli, B. subtilis and S. epidermidis (Fig. 4B).
SC
AP16-A rapidly permeabilized the membranes of B. subtilis and S. epidermidis within 5 and 20 min, respectively. By contrast, the membranes of E. coli were permeabilized within 50 min. The
with the killing kinetics (Fig. 3).
M AN U
results indicated that the membrane-permeabilizing effect of AP16-A on bacteria were consistent
As shown in Figure 4A, green fluorescence was detected within E. coli, B. subtilis and S. epidermidis. A gel retardation assay was conducted to analyze the potential interaction between AP16-A and nucleic acids (Fig. 4C). The results showed that AP16-A exhibited strong binding activity to plasmid DNA and delayed its electrophoretic mobility in a dose-dependent manner.
TE D
Plasmid DNA was completely retarded at a peptide/DNA ratio of 2. The ability of AP16-A to permeabilize the OM was determined by using the fluorescent dye NPN assay. NPN is a hydrophobic fluorescent molecule that fluoresces weakly in an aqueous
EP
environment, but fluoresces strongly in a hydrophobic environment like the outer bacterial membrane [27]. The OM of a bacterial cell is impermeable to NPN under normal conditions.
AC C
However, permeabilization of the OM by AMPs allows the NPN to enter the hydrophobic environment of the membrane, thereby leading to enhanced fluorescence in the cell. As shown in Fig. 5A, an increase in fluorescence was detected in the presence of NPN in E. coli at the concentrations of 1 µg/ml to 128 µg/ml, indicating the dose-dependent OM permeabilization of AP16-A. At a concentration of 128 µg/ml, AP16-A immediately permeabilized the OM of E. coli (Fig. 5B), and the results of other concentration of AP16-A were the same (data not shown). To determine whether LPS could be a potential target of AP16-A, different concentrations of LPS 10
ACCEPTED MANUSCRIPT
were incubated with AP16-A for 2 h. The results showed that the antimicrobial activity of AP16-A was significantly reduced by the addition of LPS at the concentration of 100 µg/ml (Fig 6). 4.
Discussion
RI PT
The accelerated emergence of microbial drug resistance poses a significant challenge to public health. The use of AMPs to solve this problem has been the focus of considerable attention [4, 28]. In this study, we designed a novel AMP (AP16-A) based on the membrane-disrupting properties
SC
of the 4E10 epitope. In order to allow initial electrostatic interaction with the negatively chanrged bacterial membrane, we added three Lys to the C-terminus of the 4E10 epitope (because the optimal charge for maximal antimicrobial activity has been shown to be +4 [29]).
M AN U
AP16-A showed excellent activity against both Gram-negative and Gram-positive bacteria. In particular, the effect of AP16-A was most potent against B. subtilis, with an MBC of 4 µg/ml, and S. epidermidis, with an MBC of 8 µg/ml. Meanwhile, the value of MBC against Gram-negative bacteria was slightly higher than that against Gram-positive bacteria. This finding can be attributed to the cell wall differences between Gram-negative and Gram-positive bacteria.
TE D
Generally, Gram-negative bacteria have a complex cell wall, with an additional outer bilayer membrane composed of LPS and phospholipids. LPS are the receptors for AMPs. By contrast, the major constituent of Gram-negative bacteria cell wall is peptidoglycan [30, 31]. Therefore, killing
EP
Gram-negative bacteria would be more difficult for AMPs, resulting in a relatively lower antimicrobial activity. These results were further confirmed by the OM permeabilization assay
AC C
and in vitro binding assay between AP16-A and LPS. After treatment with AP16-A, the OM of E. coli was immediately permeabilized. In addition, AP16-A showed strong binding activity to LPS. Compared with melittin, AP16-A was shown to be safe for mammalian cells at a relatively
high concentration. In addition, no apparent toxicity of AP16-A was observed at the 10 and 20 mg/kg doses when injected i.p. in BALB/c mice. Taken together with findings from the hemolytic assay, these results indicated that AP16-A harbors the right balance between antimicrobial activity and safety to eukaryotic cells. 11
ACCEPTED MANUSCRIPT
In this study, AP16-A was shown to kill Gram-negative bacteria (E. coli) and Gram-positive bacteria (B. subtilis and S. epidermidis) by different mechanisms. We examined the localization of AP16-A in bacteria to gain better insights into its mechanism of action. AP16-A accumulated on
RI PT
and permeabilized the membrane of bacteria before translocation inside the cells. Furthermore, AP16-A rapidly permeabilized the membranes of B. subtilis and S. epidermidis within 5 and 20 min, respectively. This result was consistent with the killing kinetics, in which B. subtilis and S.
SC
epidermidis were completely killed within 5 and 20 min, respectively. By contrast, SYTOX Green uptake experiments with Gram-negative bacteria showed a slow membrane permeabilization within 50 min. However, the complete killing of Gram-negative bacteria occurred over a period
M AN U
of 100 min. Thus, AP16-A may have interacted with other compounds after permeabilizing the membranes. The gel retardation assay confirmed that AP16-A could combine with DNA, which is the intracellular target of AMPs. Taken together, the results of this study indicated that AP16-A killed Gram-positive bacteria by membrane permeabilization as exhibited by the rapid killing kinetics; meanwhile, Gram-negative bacteria were killed by the peptide through the combined
TE D
effects of a slower membrane permeabilization process and targeting of an intracellular target (DNA), leading in the slow killing kinetics.
In conclusion, AP16-A derived from the MPER of HIV-1 exhibited broad-spectrum
EP
antimicrobial activity and selective cytotoxicity. The peptide exerted its toxic effects against Gram-negative bacteria and Gram-positive bacteria via different mechanisms. Thus, AP16-A has
AC C
the potential to be developed as a novel antimicrobial agent. Acknowledgments
The current work was supported by the National Natural Science Foundation of China (Grant No. 30700998 and 81200289), Jilin Province Science Foundation for Youths (Grant No. 20130522007JH), and Key Projects of Science and Technology Bureau of Changchun City (Grant No. 14KG052). 12
ACCEPTED MANUSCRIPT
References [1] D.I. Andersson, D. Hughes, Antibiotic resistance and its cost: is it possible to reverse resistance?, Nature reviews. Microbiology, 8 (2010) 260-271. [2] S.B. Winfred, G. Meiyazagan, J.J. Panda, V. Nagendrababu, K. Deivanayagam, V.S. Chauhan, G.
RI PT
Venkatraman, Antimicrobial activity of cationic peptides in endodontic procedures, European journal of dentistry, 8 (2014) 254-260.
[3] R.E. Hancock, H.G. Sahl, Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies, Nature biotechnology, 24 (2006) 1551-1557.
[4] S. Pathak, V.S. Chauhan, Rationale-based, de novo design of dehydrophenylalanine-containing antibiotic peptides and systematic modification in sequence for enhanced potency, Antimicrobial agents and
SC
chemotherapy, 55 (2011) 2178-2188.
[5] B.M. Peters, M.E. Shirtliff, M.A. Jabra-Rizk, Antimicrobial peptides: primeval molecules or future drugs?, PLoS pathogens, 6 (2010) e1001067.
M AN U
[6] M.N. Melo, R. Ferre, M.A. Castanho, Antimicrobial peptides: linking partition, activity and high membrane-bound concentrations, Nature reviews. Microbiology, 7 (2009) 245-250. [7] M. Zasloff, Antimicrobial peptides of multicellular organisms, Nature, 415 (2002) 389-395. [8] K.A. Brogden, Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria?, Nature reviews. Microbiology, 3 (2005) 238-250.
[9] A. Tossi, L. Sandri, A. Giangaspero, Amphipathic, alpha-helical antimicrobial peptides, Biopolymers, 55 (2000) 4-30.
TE D
[10] M.S. Diz, A.O. Carvalho, R. Rodrigues, A.G. Neves-Ferreira, M. Da Cunha, E.W. Alves, A.L. Okorokova-Facanha, M.A. Oliveira, J. Perales, O.L. Machado, V.M. Gomes, Antimicrobial peptides from chili pepper seeds causes yeast plasma membrane permeabilization and inhibits the acidification of the medium by yeast cells, Biochimica et biophysica acta, 1760 (2006) 1323-1332. [11] A.K. Marr, W.J. Gooderham, R.E. Hancock, Antibacterial peptides for therapeutic use: obstacles and
EP
realistic outlook, Current opinion in pharmacology, 6 (2006) 468-472. [12] S. Fernandez-Lopez, H.S. Kim, E.C. Choi, M. Delgado, J.R. Granja, A. Khasanov, K. Kraehenbuehl, G. Long, D.A. Weinberger, K.M. Wilcoxen, M.R. Ghadiri, Antibacterial agents based on the cyclic
AC C
D,L-alpha-peptide architecture, Nature, 412 (2001) 452-455. [13] P. Li, Y.F. Poon, W. Li, H.Y. Zhu, S.H. Yeap, Y. Cao, X. Qi, C. Zhou, M. Lamrani, R.W. Beuerman, E.T. Kang, Y. Mu, C.M. Li, M.W. Chang, S.S. Leong, M.B. Chan-Park, A polycationic antimicrobial and biocompatible hydrogel with microbe membrane suctioning ability, Nature materials, 10 (2011) 149-156. [14] R. Wyatt, J. Sodroski, The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens, Science, 280 (1998) 1884-1888. [15] S.A. Vishwanathan, E. Hunter, Importance of the membrane-perturbing properties of the membrane-proximal external region of human immunodeficiency virus type 1 gp41 to viral fusion, Journal of virology, 82 (2008) 5118-5126. 13
ACCEPTED MANUSCRIPT
[16] L. Cai, M. Gochin, K. Liu, Biochemistry and biophysics of HIV-1 gp41 - membrane interactions and implications for HIV-1 envelope protein mediated viral-cell fusion and fusion inhibitor design, Current topics in medicinal chemistry, 11 (2011) 2959-2984. [17] M.E. Selsted, M.J. Novotny, W.L. Morris, Y.Q. Tang, W. Smith, J.S. Cullor, Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils, The Journal of biological chemistry, 267 (1992)
RI PT
4292-4295.
[18] B. Deslouches, S.M. Phadke, V. Lazarevic, M. Cascio, K. Islam, R.C. Montelaro, T.A. Mietzner, De novo generation of cationic antimicrobial peptides: influence of length and tryptophan substitution on antimicrobial activity, Antimicrobial agents and chemotherapy, 49 (2005) 316-322.
[19] M. Scocchi, A. Tossi, R. Gennaro, Proline-rich antimicrobial peptides: converging to a non-lytic
SC
mechanism of action, Cellular and molecular life sciences : CMLS, 68 (2011) 2317-2330.
[20] I. Radzishevsky, M. Krugliak, H. Ginsburg, A. Mor, Antiplasmodial activity of lauryl-lysine oligomers, Antimicrobial agents and chemotherapy, 51 (2007) 1753-1759.
M AN U
[21] I.S. Radzishevsky, T. Kovachi, Y. Porat, L. Ziserman, F. Zaknoon, D. Danino, A. Mor, Structure-activity relationships of antibacterial acyl-lysine oligomers, Chemistry & biology, 15 (2008) 354-362.
[22] R.I. Lehrer, M.E. Selsted, D. Szklarek, J. Fleischmann, Antibacterial activity of microbicidal cationic proteins 1 and 2, natural peptide antibiotics of rabbit lung macrophages, Infection and immunity, 42 (1983) 10-14.
[23] M.A. Miller, R.F. Garry, J.M. Jaynes, R.C. Montelaro, A structural correlation between lentivirus transmembrane proteins and natural cytolytic peptides, AIDS research and human retroviruses, 7 (1991)
TE D
511-519.
[24] Y.C. Huang, Y.M. Lin, T.W. Chang, S.J. Wu, Y.S. Lee, M.D. Chang, C. Chen, S.H. Wu, Y.D. Liao, The flexible and clustered lysine residues of human ribonuclease 7 are critical for membrane permeability and antimicrobial activity, The Journal of biological chemistry, 282 (2007) 4626-4633. [25] C.P. Chen, J.C. Chou, B.R. Liu, M. Chang, H.J. Lee, Transfection and expression of plasmid DNA in
EP
plant cells by an arginine-rich intracellular delivery peptide without protoplast preparation, FEBS letters, 581 (2007) 1891-1897.
[26] P. Aramwit, S. Kanokpanont, T. Nakpheng, T. Srichana, The effect of sericin from various extraction
AC C
methods on cell viability and collagen production, International journal of molecular sciences, 11 (2010) 2200-2211.
[27] B. Loh, C. Grant, R.E. Hancock, Use of the fluorescent probe 1-N-phenylnaphthylamine to study the interactions of aminoglycoside antibiotics with the outer membrane of Pseudomonas aeruginosa, Antimicrobial agents and chemotherapy, 26 (1984) 546-551. [28] H.L. Chen, P.Y. Su, Y.S. Chang, S.Y. Wu, Y.D. Liao, H.M. Yu, T.L. Lauderdale, K. Chang, C. Shih, Identification of a novel antimicrobial peptide from human hepatitis B virus core protein arginine-rich domain (ARD), PLoS pathogens, 9 (2013) e1003425.
14
ACCEPTED MANUSCRIPT
[29] A. Giangaspero, L. Sandri, A. Tossi, Amphipathic alpha helical antimicrobial peptides, European journal of biochemistry / FEBS, 268 (2001) 5589-5600. [30] Y. Shai, Mode of action of membrane active antimicrobial peptides, Biopolymers, 66 (2002) 236-248. [31] A. Pini, C. Falciani, E. Mantengoli, S. Bindi, J. Brunetti, S. Iozzi, G.M. Rossolini, L. Bracci, A novel tetrabranched antimicrobial peptide that neutralizes bacterial lipopolysaccharide and prevents septic shock
RI PT
in vivo, FASEB journal : official publication of the Federation of American Societies for Experimental
AC C
EP
TE D
M AN U
SC
Biology, 24 (2010) 1015-1022.
15
ACCEPTED MANUSCRIPT
Table 1 Amino acid sequences of designed peptides Sequence
MW
Ac-NWFNITNWLWYIKKKK-NH2
2224.66
AP16-A
Ac-NWFAITNWLWYIKKKK-NH2
2181.63
AP16-D
Ac-NWFDITNWLWYIKKKK-NH2
2225.64
AP16-K
Ac-NWFKITNWLWYIKKKK-NH2
2238.73
AP16+K
Ac-NWFDITNWLWYIKKKKK-NH2
2353.82
AP16-I
Ac-NWFDITNWIWYIKKKK-NH2
2224.66
AP16-2W
Ac-NWFDITNKLWYIKKKK-NH2
2167.6
M AN U
Mutated amino acids are bolded.
SC
AP16
RI PT
Peptide
Table 2 Antimicrobial activity of peptides Peptide
MBC: µg/ml (µM)
Gram-negative bacteria
Gram-positive bacteria
B. subtilis （ATCC 6633)
S. epidermidis (ATCC 12228)
AP16
>256 ( >115.07)
16-32 （7.19-14.38）
128-256 ( 57.54-115.07)
AP16-A
8-16 ( 3.67-7.34)
2-4 ( 0.917-1.834)
4-8 ( 1.834-3.67)
AP16-D
32-64 (14.38-28.75)
8-16 ( 3.594-7.188)
64-128 ( 28.75-57.5)
AP16-K
8-16 ( 3.58-7.15)
4-8 ( 1.786-3.573)
8-16 ( 3.573-7.15)
AP16+K
64-128 ( 27.19-54.38)
8-16 ( 3.339-6.797)
32-64 ( 13.59-27.19)
AP16-I
128-256 ( 57.53-115.07)
64-128 ( 28.765-57.53)
>256 ( >115.07)
AP16-2W 128-256 ( 59.05-118.1)
128-256 ( 59.05-118.1)
>256 ( >118.1)
Melittin
2-4 (0.7-1.4)
1-2 ( 0.35-0.7)
AC C
EP
TE D
E.coli (DH5α）
4-8 ( 1.4-2.8)
Melittin was used as a reference AMP. Samples were measured in triplicate. 16
ACCEPTED MANUSCRIPT
Fig. 1 Dose-dependent killing of bacteria. Counts of bacteria (log of the number of CFU per milliliter) remaining upon treatment of (A) E. coli, (B) B. subtilis and (C) S. epidermidis are plotted as a function of the peptide concentration. Melittin was used as a control.
RI PT
Fig. 2 Toxicity of AP16-A in vitro and in vivo. The peptide at (A) 1 × MBC concentration was incubated with 4% guinea pig red blood cells for 1 h at 37°C. Survival of (B) hCMEC/D3 cells after exposure to peptides for 3 h. Melittin was used as a positive control. BALB/c mice were
SC
injected i.p. with peptide (10 and 20 mg/kg of body weight). (C) Survival and (D) weight change of mice after injection with AP16-A.
Fig. 3 Kinetics of bacterial killing. E. coli, B. subtilis and S. epidermidis were treated with
Fig. 4
M AN U
AP16-A at 1 × MBC. The surviving bacteria were measured at indicated time points. Bactericidal mechanism of AP16-A. (A) Localization of FITC-labeled AP16-A on
bacteria. (a) E. coli, (b) B. subtilis and (c) S. epidermidis were treated with AP16-A at the concentration of 0.5 × MBC. The bacteria were washed, fixed and stained with DAPI (blue) before observation by confocal fluorescence microscopy. (B) SYTOX Green uptake of E. coli, B.
TE D
subtilis and S. epidermidis by AP16-A. (C) DNA-binding activity of AP16-A. AP16-A was incubated with pET-20b DNA at various N/P ratios. Fig. 5
OM permeabilization assessment using NPN uptake assay on E. coli. (A) AP16-A at
EP
various concentrations was mixed with E. coli. Untreated cells served as the control. (B) Time course of AP16-A-mediated permeabilization of the E. coli OM at the concentration of 128
AC C
µg/ml.
Fig. 6
Dose response effects of LPS on the bacterial activity of AP16-A. LPS from
E.coli was mixed with E.coli and AP16-A (1 × MBC) for 2 h. The dotted line represents no LPS and AP16-A.
17
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Novel peptide AP16-A from membrane-proximal external region of HIV-1 is described. AP16-A shows antimicrobial activities against Gram-negative and -positive bacteria. AP16-A is safe in eukaryocytic cells.
AC C
EP
TE D
M AN U
SC
RI PT
AP16-A kills Gram-negative bacteria and -positive bacteria by different mechanisms.
S0300-9084(16)00049-3
DOI:
10.1016/j.biochi.2016.02.006
Reference:
BIOCHI 4938
To appear in:
Biochimie
Received Date: 15 December 2015 Accepted Date: 9 February 2016
Please cite this article as: X. He, H. Zhang, Y. Shi, X. Gong, S. Guan, H. Yin, L. Yang, Y. Yu, Z. Kuai, D. Liu, R. Hua, S. Wang, Y. Shan, A novel antimicrobial peptide derived from membrane-proximal external region of human immunodeficiency virus type 1, Biochimie (2016), doi: 10.1016/j.biochi.2016.02.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
ABSTRACT With increasing microbial drug resistance worldwide, antimicrobial peptides (AMPs) are considered promising alternatives to addressing this problem. In this study, a series of synthetic
RI PT
peptides were designed based on the membrane-disrupting properties of the membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) envelope protein. The peptide AP16-A was found to exhibit the most effective antimicrobial activities against both
SC
Gram-negative and Gram-positive bacteria. The minimal bactericidal concentration (MBC) of AP16-A ranged from 2 µg/ml to 16 µg/ml. AP16-A had no detectable cytotoxicity in various tissue cultures and a mouse model. Furthermore, results of confocal fluorescence microscopy and
M AN U
the SYTOX Green uptake assay indicated that AP16-A killed Gram-negative bacteria by the combined effects of relatively slow membrane permeabilization and interaction with an intracellular target, while it killed Gram-positive bacteria by a fast membrane permeabilization process, which achieved relatively more rapid bacterial killing kinetics. The results of this study support the potential use of AP16-A as an AMP.
AC C
EP
killing kinetics
TE D
Keywords: antimicrobial peptides, antimicrobial activity, membrane permeabilization, bacterial
1
ACCEPTED MANUSCRIPT
A novel antimicrobial peptide derived from membrane-proximal
RI PT
external region of human immunodeficiency virus type 1
Xiaoqiu Hea, Huayan Zhanga, Yuhua Shia, Xin Gonga, Shanshan Guana, He Yina, Lan Yanga, Yongjiao Yua, Ziyu Kuaia, Dongni Liua, Rui Huab, Song Wangc, and Yaming Shana,d*
National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University,
Changchun, Jilin, China;
b
SC
a
Department of Hepatology, The First Hospital, Jilin University,
Changchun, Jilin, China; c State Key Laboratory of Theoretical and Computational Chemistry,
M AN U
Institute of Theoretical Chemistry, Jilin University, Changchun, Jilin, China; d Key Laboratory for Molecular Enzymology and Engineering, The Ministry of Education, School of Life Sciences, Jilin University, Changchun, Jilin, China
EP
TE D
*Address correspondence to Yaming Shan, [email protected]
AC C
Abbreviations: AMPs: antimicrobial peptides, MPER: membrane-proximal external region, HIV-1: human immunodeficiency virus type 1, LPS: lipopolysaccharides, CFU: colony-forming units, MBC: minimal bactericidal concentration, OM: outer membrane
1
ACCEPTED MANUSCRIPT
ABSTRACT With increasing microbial drug resistance worldwide, antimicrobial peptides (AMPs) are considered promising alternatives to addressing this problem. In this study, a series of synthetic
RI PT
peptides were designed based on the membrane-disrupting properties of the membrane-proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) envelope protein. The peptide AP16-A was found to exhibit the most effective antimicrobial activities against both
SC
Gram-negative and Gram-positive bacteria. The minimal bactericidal concentration (MBC) of AP16-A ranged from 2 µg/ml to 16 µg/ml. AP16-A had no detectable cytotoxicity in various tissue cultures and a mouse model. Furthermore, results of confocal fluorescence microscopy and
M AN U
the SYTOX Green uptake assay indicated that AP16-A killed Gram-negative bacteria by the combined effects of relatively slow membrane permeabilization and interaction with an intracellular target, while it killed Gram-positive bacteria by a fast membrane permeabilization process, which achieved relatively more rapid bacterial killing kinetics. The results of this study support the potential use of AP16-A as an AMP.
AC C
EP
killing kinetics
TE D
Keywords: antimicrobial peptides, antimicrobial activity, membrane permeabilization, bacterial
2
ACCEPTED MANUSCRIPT
1.
Introduction The accelerated emergence of drug-resistant bacterial strains caused by the extensive use of
traditional antibiotics is a current major threat [1, 2]. Therefore, the development of a more
RI PT
effective treatment to overcome the problem of drug resistance is urgently needed. Antimicrobial peptides (AMPs) can provide a possible alternative to traditional drugs with a new mode of action and remarkable therapeutic effects [3, 4]. Generally, naturally occurring AMPs contain 12–50
SC
amino acids in length, with an overall positive charge and an amphipathic structure. Most AMPs can directly bind to the bacterial membrane and kill the organism by accessing intracellular targets through membrane permeabilization [5-8]. The net positive charge facilitates the
M AN U
interaction between AMPs and negatively charged microbial surfaces, with the amphipathic secondary structure allowing AMPs to incorporate into microbial membranes [9]. To date, more than 1,000 AMPs have been identified in various species, including plants, insects, fish, frogs and mammals [10]. However, most natural AMPs are large, have low potency and are toxic to host cells [11]. Considering these complications, different synthetic AMPs, as well as corresponding
TE D
mimics, have been the focus of considerable interest in studies aimed at increasing the effectiveness of AMPs [12, 13]. Short designer AMPs that are less likely to induce resistance and
[4].
EP
show low toxicity to host cells and tissues have the potential to be the most effective candidates
The essential process of HIV-1 entry into a cell is the fusion of the viral and target cell
AC C
membranes. This crucial step is mediated by the HIV-1 envelope protein [14]. The membrane-proximal external region (MPER, Ac-665WASLWNWFNITNW LWYIK683-NH2), a highly conserved domain of the HIV-1 gp41 ectodomain, plays a critical role in viral envelope glycoprotein mediated-fusion and infectivity [15, 16]. In addition, the epitopes of well-characterized neutralizing antibodies 2F5, Z13 and 4E10 overlap this conserved region. Previous reports have shown that the MPER, with an unusually high percentage of tryptophan (Trp) residues, likely contributes to the membrane-disrupting properties [15]. Indolicidin, an 3
ACCEPTED MANUSCRIPT
analog of MPER, is also capable of disrupting membranes and has an unusually high number of Trps as well. Indolicidin is 13 amino acids (ILPWKWPWWPWRR) in length and is a naturally occurring AMP isolated from bovine neutrophils [17]. However, a high Trp content in AMPs is
RI PT
associated with high hemolysis, making indolicidin unsuitable for use as an antimicrobial agent [18]. Based on the membrane-disrupting properties of the MPER, we hypothesized that synthetic peptides derived from this region, such as the 4E10 epitope (Ac-NWFNITNWLWYIK-NH2), may
SC
have functional properties similar to those of known AMPs.
Although the sequences of AMPs vary greatly, certain amino acids, such as lysine (Lys) or arginine (Arg), are key components of AMPs [19]. In particular, Lys-rich AMPs exhibit rapid,
M AN U
nonhemolytic, broad-spectrum microbicidal properties [20, 21]. Thus, we aimed to increase this activity by adding Lys to the C-terminus of the 4E10 epitope to generate an AMP with 16 amino acids (AP16: Ac-NWFNITNWLWYIKKKK-NH2). In this study, a series of mutant peptides of AP16 were synthesized, and their antimicrobial activities in vitro against Gram-negative and Gram-positive bacteria were determined. Among them, AP16-A exhibited the highest
TE D
antimicrobial activity. Potential mechanisms for bactericidal activity were also investigated. By showing low toxicity in eukaryocytes and hemolytic activity, AP16-A has the potential for further development and use as an antibiotic. Materials and methods
EP
2.
2.1 Bacteria and reagents
AC C
Escherichia coli DH5α was purchased from Takara. Bacillus subtilis ATCC 6633 and Staphylococcus epidermidis ATCC 12228 were purchased from the American Type CultureCollection
(ATCC).
Melittin,
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium
bromide (MTT) and lipopolysaccharides (LPS) were purchased from Sigma-Aldrich. Poly-L-lysine and 4,6-diamidino-2-phenylindole (DAPI) were purchased from Roster. SYTOX Green were purchased from KeyGEN BioTECH. N-phenyl-1-napthylamine (NPN) was purchased from Aladdin. 4
ACCEPTED MANUSCRIPT
2.2 Animals and ethics statement
Four weeks old BALB/c female mice(18−20 g), were purchased from the Changchun Institute of Biological Products Co., Ltd. The animal trials in this study were carried out
RI PT
in accordance with the Regulations and the Administration of Affairs Concerning
Experimental Animals approved by the State Council of People’s Republic of China (11-14-1988). The Institutional Animal Care and Use Committee (IACUC) of Jilin
SC
University approved all animal procedures (permit number: SCXK 2013-0001). 2.3 Peptides
All peptides used in this study (listed in Table 1) were synthesized by the Chinese Peptide
M AN U
Company (Hangzhou, China). Vendors provided data on peptide characterizations, including high-performance liquid chromatography (>98% purity) and mass spectrometry. All lyophilized peptides were dissolved in water to 1 mg/ml stocks and stored at −20°C. 2.4 Antimicrobial activity
Antimicrobial activity was determined using a method similar to that described previously
TE D
[22-24]. Bacterial suspensions were cultured to mid-logarithmic phase at 37°C and washed by several cycles of centrifugation and resuspension in 10 mM potassium phosphate buffer (PB), pH 7.2. Bacteria were diluted to between 1 × 106 colony-forming units (CFU)/ml and 2 × 106 CFU/ml
EP
in PB and then treated in the assay. Peptides were diluted two-fold in 96-well plates in the same buffer. One hundred microliters of bacteria was mixed with 100 µl of peptides, followed by
AC C
incubation at 37°C for 2 h without shaking. At the end of the incubation period, a 100 µl aliquot from each dilution was plated on tryptic soy agar and allowed to grow at 37°C overnight for the measurement of the minimal bactericidal concentration (MBC). The MBC is defined as the lowest peptide concentration on the agar plate, which exhibited no bacterial growth (zero colony). All peptides were tested in triplicate. 2.5 Dose-dependent bacterial killing 5
ACCEPTED MANUSCRIPT
Bacteria and peptides were prepared as in the antimicrobial activity assay. Serial 10-fold dilutions of control and test wells were included. A 100 µl aliquot from each dilution was plated on tryptic soy agar and incubated overnight at 37°C. Colonies of surviving bacteria were counted
RI PT
to determine the extent of peptide-induced killing under each concentration of peptides. 2.6 Hemolytic assay
The hemolytic activities of peptides were determined by hemolysis of guinea pig red blood
SC
cells. Freshly collected heparinized guinea pig blood was centrifuged at 1000 × g for 30 min. The erythrocytes were washed two times with saline and centrifuged again at 1000 × g for 10 min. A solution of 4% (v/v) guinea pig red blood cells was prepared and mixed with serial dilutions of
M AN U
peptides in saline in 96-well plates. The mixture was then incubated for 1 h at 37°C. After centrifugation at 1000 × g for 30 min, the percentage of hemolysis was determined by measuring the absorbance of the supernatant at a wavelength of 450 nm. Cells incubated with saline alone served as the negative control, and guinea pig red blood cell sample lysed with 1% Triton X-100
2.7 In vitro cytotoxicity
TE D
was used to represent 100% lysis (positive control).
The cytotoxicity of the peptides to hCMEC/D3 cells was measured by the MTT assay. Briefly, cells were seeded at 104 cells/well in 96-well plates at 37°C overnight. The next day, serial
EP
dilutions of peptides were added to each well. The untreated cells served as the negative control, and melittin, the major toxin of bee venom, was used as a positive control [25]. After 3 h of
AC C
incubation, 10% MTT solution (5 mg/ml) in phosphate-buffered saline was added, and the plate was incubated for 2 h with 5% CO2 at 37°C. The supernatant was then removed, and 150 µl dimethyl sulfoxide was added. The absorbance of the supernatant at a wavelength of 490 nm was measured.
2.8 In vivo cytotoxicity
BALB/c female mice were injected intraperitoneally (i.p.) with AP16-A (10 and 20 mg/kg of body weight) in saline to test the acute toxicity of this peptide in vivo. Peptides 6
ACCEPTED MANUSCRIPT
were administered in 200 µl saline, and the control were injected i.p. with an equal volume of saline. Each group consisted of six mice. After peptide injection, the number of dead mice and the change in body weight were recorded daily for 7 days
RI PT
post-injection. 2.9 Killing kinetics
Bacteria (1 × 106 CFU/ml to 2 × 106 CFU/ml) were treated with peptides at the MBC in PB. At
SC
the indicated time, aliquots of this mixture were serially diluted with Luria broth, which substantially inhibited the killing process, and then placed on tryptic soy agar plates and
of peptide exposure time. 2.10 Confocal fluorescence microscopy
M AN U
incubated overnight for viability measurement. The number of colonies was plotted as a function
The cellular localization of AP16-A was observed by confocal fluorescence microscopy. Bacteria were grown to the mid-logarithmic phase and then harvested, washed and resuspended in PB to a concentration of approximately 108 CFU/ml. The suspension was mixed with fluorescein
TE D
isothiocyanate (FITC)-labeled AP16-A solution to yield a final peptide concentration corresponding to 0.5 × MBC. Following incubation at 37°C, the cells were harvested by centrifugation, washed with PB and smeared on a poly-L-lysine-coated slide. Localization of
EP
labeled peptide was monitored under an inverted confocal microscope equipped with a 63× oil immersion lens (CarlZeiss LSM 710).
AC C
2.11 SYTOX Green uptake assay
Briefly, bacterial suspensions of 107 CFU/ml were prepared and mixed with 1 µM SYTOX Green for 15 min in the dark. After the addition of AP16-A to the final concentration corresponding to the MBC, uptake of SYTOX Green was determined using a fluorescence spectrophotometer (filter wavelengths of 485 and 520 nm for excitation and emission, respectively). 2.12 Gel retardation assay 7
ACCEPTED MANUSCRIPT
Briefly, AP16-A was incubated with pET-20b plasmid DNA at different N/P ratios (0, 0.2, 0.4, 0.6, 0.8, 1, 2 and 3) for 30 min at 37°C to form peptide/DNA complexes. The N/P ratio was defined as the proportion of amino nitrogen (NH3+) of AP16-A relative to the phosphate (PO4−) of
2.13 Outer membrane (OM) permeabilization assay
RI PT
DNA [26]. These complexes were analyzed by electrophoresis on a 1% agarose gel in TAE.
The membrane-permeabilizing activity of the peptide was determined by using the fluorescent
SC
dye NPN assay, as described previously [27]. Bacterial suspensions of 107 CFU/ml were prepared and mixed with 10 µM NPN, and uptake of the dye was determined using a fluorescence spectrophotometer (filter wavelengths of 350 and 420 nm for excitation and emission,
M AN U
respectively). The increase in fluorescence due to partitioning of NPN uptake into the OM was determined by increasing the concentration of the peptide. All experiments were conducted three times.
2.14 Assay of in vitro binding between AP16-A and LPS
Bacteria were prepared as for the antimicrobial activity assay. LPS from E. coli was
TE D
mixed with E. coli and AP16-A (1× MBC) for 2 hours. Serial 10-fold dilutions of control and test wells were included. A 100 µl aliquot from each dilution was plated on tryptic soy agar and incubated overnight at 37°C. Colonies of surviving bacteria were counted to
3.
Results
EP
determine the peptide-induced killing after adding LPS.
AC C
3.1. Antimicrobial activity
The antimicrobial activities of AP16 and its analogs against E. coli, B. subtilis, and S. epidermidis are shown in Table 2. AP16-A showed high broad-spectrum antimicrobial activity against both Gram-negative and Gram-positive bacteria. The MBCs of AP16-A were lower than 4 µg/ml for B. subtilis and 8 µg/ml for S. epidermidis. E. coli was the least sensitive to AP16-A (MBC = 16 µg/ml). As indicated in Fig. 1, bacterial killing by AP16-A was dose-dependent, further validating its antimicrobial activity. AP16-A exhibited more than a 2 log reduction in 8
ACCEPTED MANUSCRIPT
colony counts (>99% of bacteria killed) for E. coli and S. epidermidis at one half of the MBC. For B. subtilis, AP16-A exhibited more than a 4 log reduction in colony counts (>99.99% of bacteria killed) at one half of the MBC and 2 log reduction in colony counts (>99% of bacteria killed) at
RI PT
one fourth of the MBC. 3.2. In vitro and in vivo cytotoxicities
Several AMPs exhibited cytolytic activities. As shown in Fig. 2A, no evident hemolysis by
SC
AP16-A was observed at a final peptide concentration corresponding to 1 × MBC for all bacteria. However, melittin was approximately 72% hemolytic for the MBC of S. epidermidis and approximately 100% hemolytic for the MBC of E. coli and B. subtilis. The standard MTT assay
M AN U
was conducted to determine the cytotoxicity of AMPs to human cells (hCMEC/D3). As shown in Fig. 2B, the concentration of peptides ranged from 1 µg/ml to 128 µg/ml. The cells treated with melittin were completely killed at a low dose (8 µg/ml). By contrast, AP16-A caused only a low level of cytotoxicity at the concentration of 128 µg/ml. Female mice were injected i.p. with AP16-A (10 and 20 mg/kg of body weight) to determine the cytotoxicity of AP16-A in the animal
TE D
model. All mice were alive after seven days (Fig. 2C), and the weight of all mice were apparently unchanged compared with the control (Fig. 2D). 3.3. Kinetics of antimicrobial activity
EP
The tested bacteria were treated with AP16-A at concentrations corresponding to the MBC to determine the time course of bacterial killing (Table 1). Results showed that killing of B. subtilis
AC C
and S. epidermidis was more rapid than E. coli. B. subtilis was immediately killed within 5 min, and S. epidermidis was killed within 20 min upon the addition of AP16-A. However, complete killing of E. coli, a Gram-negative bacteria, by AP16-A occurred over the course of 100 min (Fig. 3).
3.4. Bactericidal mechanisms of AP16-A The localization of peptides in bacteria was examined by using FITC-labeled analogs corresponding to 0.5 × MBC to gain insight into the bactericidal mechanisms of AP16-A. Upon 9
ACCEPTED MANUSCRIPT
peptide treatment, the green fluorescence (FITC-labeled AP16-A) surrounded the blue fluorescence (nucleic acids), indicating that AP16-A accumulated on the membrane (Fig. 4A). The SYTOX Green uptake assay was conducted to further examine the effect of AP16-A on
RI PT
membranes. SYTOX Green is a membrane-impermeable nucleic acid stain that enters only membrane-disrupted cells. The results showed that a significant degree of membrane permeabilization was induced by the peptide on E. coli, B. subtilis and S. epidermidis (Fig. 4B).
SC
AP16-A rapidly permeabilized the membranes of B. subtilis and S. epidermidis within 5 and 20 min, respectively. By contrast, the membranes of E. coli were permeabilized within 50 min. The
with the killing kinetics (Fig. 3).
M AN U
results indicated that the membrane-permeabilizing effect of AP16-A on bacteria were consistent
As shown in Figure 4A, green fluorescence was detected within E. coli, B. subtilis and S. epidermidis. A gel retardation assay was conducted to analyze the potential interaction between AP16-A and nucleic acids (Fig. 4C). The results showed that AP16-A exhibited strong binding activity to plasmid DNA and delayed its electrophoretic mobility in a dose-dependent manner.
TE D
Plasmid DNA was completely retarded at a peptide/DNA ratio of 2. The ability of AP16-A to permeabilize the OM was determined by using the fluorescent dye NPN assay. NPN is a hydrophobic fluorescent molecule that fluoresces weakly in an aqueous
EP
environment, but fluoresces strongly in a hydrophobic environment like the outer bacterial membrane [27]. The OM of a bacterial cell is impermeable to NPN under normal conditions.
AC C
However, permeabilization of the OM by AMPs allows the NPN to enter the hydrophobic environment of the membrane, thereby leading to enhanced fluorescence in the cell. As shown in Fig. 5A, an increase in fluorescence was detected in the presence of NPN in E. coli at the concentrations of 1 µg/ml to 128 µg/ml, indicating the dose-dependent OM permeabilization of AP16-A. At a concentration of 128 µg/ml, AP16-A immediately permeabilized the OM of E. coli (Fig. 5B), and the results of other concentration of AP16-A were the same (data not shown). To determine whether LPS could be a potential target of AP16-A, different concentrations of LPS 10
ACCEPTED MANUSCRIPT
were incubated with AP16-A for 2 h. The results showed that the antimicrobial activity of AP16-A was significantly reduced by the addition of LPS at the concentration of 100 µg/ml (Fig 6). 4.
Discussion
RI PT
The accelerated emergence of microbial drug resistance poses a significant challenge to public health. The use of AMPs to solve this problem has been the focus of considerable attention [4, 28]. In this study, we designed a novel AMP (AP16-A) based on the membrane-disrupting properties
SC
of the 4E10 epitope. In order to allow initial electrostatic interaction with the negatively chanrged bacterial membrane, we added three Lys to the C-terminus of the 4E10 epitope (because the optimal charge for maximal antimicrobial activity has been shown to be +4 [29]).
M AN U
AP16-A showed excellent activity against both Gram-negative and Gram-positive bacteria. In particular, the effect of AP16-A was most potent against B. subtilis, with an MBC of 4 µg/ml, and S. epidermidis, with an MBC of 8 µg/ml. Meanwhile, the value of MBC against Gram-negative bacteria was slightly higher than that against Gram-positive bacteria. This finding can be attributed to the cell wall differences between Gram-negative and Gram-positive bacteria.
TE D
Generally, Gram-negative bacteria have a complex cell wall, with an additional outer bilayer membrane composed of LPS and phospholipids. LPS are the receptors for AMPs. By contrast, the major constituent of Gram-negative bacteria cell wall is peptidoglycan [30, 31]. Therefore, killing
EP
Gram-negative bacteria would be more difficult for AMPs, resulting in a relatively lower antimicrobial activity. These results were further confirmed by the OM permeabilization assay
AC C
and in vitro binding assay between AP16-A and LPS. After treatment with AP16-A, the OM of E. coli was immediately permeabilized. In addition, AP16-A showed strong binding activity to LPS. Compared with melittin, AP16-A was shown to be safe for mammalian cells at a relatively
high concentration. In addition, no apparent toxicity of AP16-A was observed at the 10 and 20 mg/kg doses when injected i.p. in BALB/c mice. Taken together with findings from the hemolytic assay, these results indicated that AP16-A harbors the right balance between antimicrobial activity and safety to eukaryotic cells. 11
ACCEPTED MANUSCRIPT
In this study, AP16-A was shown to kill Gram-negative bacteria (E. coli) and Gram-positive bacteria (B. subtilis and S. epidermidis) by different mechanisms. We examined the localization of AP16-A in bacteria to gain better insights into its mechanism of action. AP16-A accumulated on
RI PT
and permeabilized the membrane of bacteria before translocation inside the cells. Furthermore, AP16-A rapidly permeabilized the membranes of B. subtilis and S. epidermidis within 5 and 20 min, respectively. This result was consistent with the killing kinetics, in which B. subtilis and S.
SC
epidermidis were completely killed within 5 and 20 min, respectively. By contrast, SYTOX Green uptake experiments with Gram-negative bacteria showed a slow membrane permeabilization within 50 min. However, the complete killing of Gram-negative bacteria occurred over a period
M AN U
of 100 min. Thus, AP16-A may have interacted with other compounds after permeabilizing the membranes. The gel retardation assay confirmed that AP16-A could combine with DNA, which is the intracellular target of AMPs. Taken together, the results of this study indicated that AP16-A killed Gram-positive bacteria by membrane permeabilization as exhibited by the rapid killing kinetics; meanwhile, Gram-negative bacteria were killed by the peptide through the combined
TE D
effects of a slower membrane permeabilization process and targeting of an intracellular target (DNA), leading in the slow killing kinetics.
In conclusion, AP16-A derived from the MPER of HIV-1 exhibited broad-spectrum
EP
antimicrobial activity and selective cytotoxicity. The peptide exerted its toxic effects against Gram-negative bacteria and Gram-positive bacteria via different mechanisms. Thus, AP16-A has
AC C
the potential to be developed as a novel antimicrobial agent. Acknowledgments
The current work was supported by the National Natural Science Foundation of China (Grant No. 30700998 and 81200289), Jilin Province Science Foundation for Youths (Grant No. 20130522007JH), and Key Projects of Science and Technology Bureau of Changchun City (Grant No. 14KG052). 12
ACCEPTED MANUSCRIPT
References [1] D.I. Andersson, D. Hughes, Antibiotic resistance and its cost: is it possible to reverse resistance?, Nature reviews. Microbiology, 8 (2010) 260-271. [2] S.B. Winfred, G. Meiyazagan, J.J. Panda, V. Nagendrababu, K. Deivanayagam, V.S. Chauhan, G.
RI PT
Venkatraman, Antimicrobial activity of cationic peptides in endodontic procedures, European journal of dentistry, 8 (2014) 254-260.
[3] R.E. Hancock, H.G. Sahl, Antimicrobial and host-defense peptides as new anti-infective therapeutic strategies, Nature biotechnology, 24 (2006) 1551-1557.
[4] S. Pathak, V.S. Chauhan, Rationale-based, de novo design of dehydrophenylalanine-containing antibiotic peptides and systematic modification in sequence for enhanced potency, Antimicrobial agents and
SC
chemotherapy, 55 (2011) 2178-2188.
[5] B.M. Peters, M.E. Shirtliff, M.A. Jabra-Rizk, Antimicrobial peptides: primeval molecules or future drugs?, PLoS pathogens, 6 (2010) e1001067.
M AN U
[6] M.N. Melo, R. Ferre, M.A. Castanho, Antimicrobial peptides: linking partition, activity and high membrane-bound concentrations, Nature reviews. Microbiology, 7 (2009) 245-250. [7] M. Zasloff, Antimicrobial peptides of multicellular organisms, Nature, 415 (2002) 389-395. [8] K.A. Brogden, Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria?, Nature reviews. Microbiology, 3 (2005) 238-250.
[9] A. Tossi, L. Sandri, A. Giangaspero, Amphipathic, alpha-helical antimicrobial peptides, Biopolymers, 55 (2000) 4-30.
TE D
[10] M.S. Diz, A.O. Carvalho, R. Rodrigues, A.G. Neves-Ferreira, M. Da Cunha, E.W. Alves, A.L. Okorokova-Facanha, M.A. Oliveira, J. Perales, O.L. Machado, V.M. Gomes, Antimicrobial peptides from chili pepper seeds causes yeast plasma membrane permeabilization and inhibits the acidification of the medium by yeast cells, Biochimica et biophysica acta, 1760 (2006) 1323-1332. [11] A.K. Marr, W.J. Gooderham, R.E. Hancock, Antibacterial peptides for therapeutic use: obstacles and
EP
realistic outlook, Current opinion in pharmacology, 6 (2006) 468-472. [12] S. Fernandez-Lopez, H.S. Kim, E.C. Choi, M. Delgado, J.R. Granja, A. Khasanov, K. Kraehenbuehl, G. Long, D.A. Weinberger, K.M. Wilcoxen, M.R. Ghadiri, Antibacterial agents based on the cyclic
AC C
D,L-alpha-peptide architecture, Nature, 412 (2001) 452-455. [13] P. Li, Y.F. Poon, W. Li, H.Y. Zhu, S.H. Yeap, Y. Cao, X. Qi, C. Zhou, M. Lamrani, R.W. Beuerman, E.T. Kang, Y. Mu, C.M. Li, M.W. Chang, S.S. Leong, M.B. Chan-Park, A polycationic antimicrobial and biocompatible hydrogel with microbe membrane suctioning ability, Nature materials, 10 (2011) 149-156. [14] R. Wyatt, J. Sodroski, The HIV-1 envelope glycoproteins: fusogens, antigens, and immunogens, Science, 280 (1998) 1884-1888. [15] S.A. Vishwanathan, E. Hunter, Importance of the membrane-perturbing properties of the membrane-proximal external region of human immunodeficiency virus type 1 gp41 to viral fusion, Journal of virology, 82 (2008) 5118-5126. 13
ACCEPTED MANUSCRIPT
[16] L. Cai, M. Gochin, K. Liu, Biochemistry and biophysics of HIV-1 gp41 - membrane interactions and implications for HIV-1 envelope protein mediated viral-cell fusion and fusion inhibitor design, Current topics in medicinal chemistry, 11 (2011) 2959-2984. [17] M.E. Selsted, M.J. Novotny, W.L. Morris, Y.Q. Tang, W. Smith, J.S. Cullor, Indolicidin, a novel bactericidal tridecapeptide amide from neutrophils, The Journal of biological chemistry, 267 (1992)
RI PT
4292-4295.
[18] B. Deslouches, S.M. Phadke, V. Lazarevic, M. Cascio, K. Islam, R.C. Montelaro, T.A. Mietzner, De novo generation of cationic antimicrobial peptides: influence of length and tryptophan substitution on antimicrobial activity, Antimicrobial agents and chemotherapy, 49 (2005) 316-322.
[19] M. Scocchi, A. Tossi, R. Gennaro, Proline-rich antimicrobial peptides: converging to a non-lytic
SC
mechanism of action, Cellular and molecular life sciences : CMLS, 68 (2011) 2317-2330.
[20] I. Radzishevsky, M. Krugliak, H. Ginsburg, A. Mor, Antiplasmodial activity of lauryl-lysine oligomers, Antimicrobial agents and chemotherapy, 51 (2007) 1753-1759.
M AN U
[21] I.S. Radzishevsky, T. Kovachi, Y. Porat, L. Ziserman, F. Zaknoon, D. Danino, A. Mor, Structure-activity relationships of antibacterial acyl-lysine oligomers, Chemistry & biology, 15 (2008) 354-362.
[22] R.I. Lehrer, M.E. Selsted, D. Szklarek, J. Fleischmann, Antibacterial activity of microbicidal cationic proteins 1 and 2, natural peptide antibiotics of rabbit lung macrophages, Infection and immunity, 42 (1983) 10-14.
[23] M.A. Miller, R.F. Garry, J.M. Jaynes, R.C. Montelaro, A structural correlation between lentivirus transmembrane proteins and natural cytolytic peptides, AIDS research and human retroviruses, 7 (1991)
TE D
511-519.
[24] Y.C. Huang, Y.M. Lin, T.W. Chang, S.J. Wu, Y.S. Lee, M.D. Chang, C. Chen, S.H. Wu, Y.D. Liao, The flexible and clustered lysine residues of human ribonuclease 7 are critical for membrane permeability and antimicrobial activity, The Journal of biological chemistry, 282 (2007) 4626-4633. [25] C.P. Chen, J.C. Chou, B.R. Liu, M. Chang, H.J. Lee, Transfection and expression of plasmid DNA in
EP
plant cells by an arginine-rich intracellular delivery peptide without protoplast preparation, FEBS letters, 581 (2007) 1891-1897.
[26] P. Aramwit, S. Kanokpanont, T. Nakpheng, T. Srichana, The effect of sericin from various extraction
AC C
methods on cell viability and collagen production, International journal of molecular sciences, 11 (2010) 2200-2211.
[27] B. Loh, C. Grant, R.E. Hancock, Use of the fluorescent probe 1-N-phenylnaphthylamine to study the interactions of aminoglycoside antibiotics with the outer membrane of Pseudomonas aeruginosa, Antimicrobial agents and chemotherapy, 26 (1984) 546-551. [28] H.L. Chen, P.Y. Su, Y.S. Chang, S.Y. Wu, Y.D. Liao, H.M. Yu, T.L. Lauderdale, K. Chang, C. Shih, Identification of a novel antimicrobial peptide from human hepatitis B virus core protein arginine-rich domain (ARD), PLoS pathogens, 9 (2013) e1003425.
14
ACCEPTED MANUSCRIPT
[29] A. Giangaspero, L. Sandri, A. Tossi, Amphipathic alpha helical antimicrobial peptides, European journal of biochemistry / FEBS, 268 (2001) 5589-5600. [30] Y. Shai, Mode of action of membrane active antimicrobial peptides, Biopolymers, 66 (2002) 236-248. [31] A. Pini, C. Falciani, E. Mantengoli, S. Bindi, J. Brunetti, S. Iozzi, G.M. Rossolini, L. Bracci, A novel tetrabranched antimicrobial peptide that neutralizes bacterial lipopolysaccharide and prevents septic shock
RI PT
in vivo, FASEB journal : official publication of the Federation of American Societies for Experimental
AC C
EP
TE D
M AN U
SC
Biology, 24 (2010) 1015-1022.
15
ACCEPTED MANUSCRIPT
Table 1 Amino acid sequences of designed peptides Sequence
MW
Ac-NWFNITNWLWYIKKKK-NH2
2224.66
AP16-A
Ac-NWFAITNWLWYIKKKK-NH2
2181.63
AP16-D
Ac-NWFDITNWLWYIKKKK-NH2
2225.64
AP16-K
Ac-NWFKITNWLWYIKKKK-NH2
2238.73
AP16+K
Ac-NWFDITNWLWYIKKKKK-NH2
2353.82
AP16-I
Ac-NWFDITNWIWYIKKKK-NH2
2224.66
AP16-2W
Ac-NWFDITNKLWYIKKKK-NH2
2167.6
M AN U
Mutated amino acids are bolded.
SC
AP16
RI PT
Peptide
Table 2 Antimicrobial activity of peptides Peptide
MBC: µg/ml (µM)
Gram-negative bacteria
Gram-positive bacteria
B. subtilis （ATCC 6633)
S. epidermidis (ATCC 12228)
AP16
>256 ( >115.07)
16-32 （7.19-14.38）
128-256 ( 57.54-115.07)
AP16-A
8-16 ( 3.67-7.34)
2-4 ( 0.917-1.834)
4-8 ( 1.834-3.67)
AP16-D
32-64 (14.38-28.75)
8-16 ( 3.594-7.188)
64-128 ( 28.75-57.5)
AP16-K
8-16 ( 3.58-7.15)
4-8 ( 1.786-3.573)
8-16 ( 3.573-7.15)
AP16+K
64-128 ( 27.19-54.38)
8-16 ( 3.339-6.797)
32-64 ( 13.59-27.19)
AP16-I
128-256 ( 57.53-115.07)
64-128 ( 28.765-57.53)
>256 ( >115.07)
AP16-2W 128-256 ( 59.05-118.1)
128-256 ( 59.05-118.1)
>256 ( >118.1)
Melittin
2-4 (0.7-1.4)
1-2 ( 0.35-0.7)
AC C
EP
TE D
E.coli (DH5α）
4-8 ( 1.4-2.8)
Melittin was used as a reference AMP. Samples were measured in triplicate. 16
ACCEPTED MANUSCRIPT
Fig. 1 Dose-dependent killing of bacteria. Counts of bacteria (log of the number of CFU per milliliter) remaining upon treatment of (A) E. coli, (B) B. subtilis and (C) S. epidermidis are plotted as a function of the peptide concentration. Melittin was used as a control.
RI PT
Fig. 2 Toxicity of AP16-A in vitro and in vivo. The peptide at (A) 1 × MBC concentration was incubated with 4% guinea pig red blood cells for 1 h at 37°C. Survival of (B) hCMEC/D3 cells after exposure to peptides for 3 h. Melittin was used as a positive control. BALB/c mice were
SC
injected i.p. with peptide (10 and 20 mg/kg of body weight). (C) Survival and (D) weight change of mice after injection with AP16-A.
Fig. 3 Kinetics of bacterial killing. E. coli, B. subtilis and S. epidermidis were treated with
Fig. 4
M AN U
AP16-A at 1 × MBC. The surviving bacteria were measured at indicated time points. Bactericidal mechanism of AP16-A. (A) Localization of FITC-labeled AP16-A on
bacteria. (a) E. coli, (b) B. subtilis and (c) S. epidermidis were treated with AP16-A at the concentration of 0.5 × MBC. The bacteria were washed, fixed and stained with DAPI (blue) before observation by confocal fluorescence microscopy. (B) SYTOX Green uptake of E. coli, B.
TE D
subtilis and S. epidermidis by AP16-A. (C) DNA-binding activity of AP16-A. AP16-A was incubated with pET-20b DNA at various N/P ratios. Fig. 5
OM permeabilization assessment using NPN uptake assay on E. coli. (A) AP16-A at
EP
various concentrations was mixed with E. coli. Untreated cells served as the control. (B) Time course of AP16-A-mediated permeabilization of the E. coli OM at the concentration of 128
AC C
µg/ml.
Fig. 6
Dose response effects of LPS on the bacterial activity of AP16-A. LPS from
E.coli was mixed with E.coli and AP16-A (1 × MBC) for 2 h. The dotted line represents no LPS and AP16-A.
17
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
AC C
EP
TE D
M AN U
SC
RI PT
ACCEPTED MANUSCRIPT
ACCEPTED MANUSCRIPT Novel peptide AP16-A from membrane-proximal external region of HIV-1 is described. AP16-A shows antimicrobial activities against Gram-negative and -positive bacteria. AP16-A is safe in eukaryocytic cells.
AC C
EP
TE D
M AN U
SC
RI PT
AP16-A kills Gram-negative bacteria and -positive bacteria by different mechanisms.