Research paperA novel antimicrobial peptide derived from membrane-proximal external region of human immunodeficiency virus type 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 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 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], [6], [7], [8]. The net positive charge facilitates the 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 1000 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 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 show low toxicity to host cells and tissues have the potential to be the most effective candidates [4].
The essential process of HIV-1 entry into a cell is the fusion of the viral and target cell 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 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 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 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, 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 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.
Section snippets
Bacteria and reagents
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
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
Discussion
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 of the 4E10 epitope. In order to allow initial electrostatic interaction with the negatively charged bacterial membrane, we added three Lys to the C-terminus of the 4E10 epitope (because the optimal charge for
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).
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