Synergistic effect of the pro-apoptosis peptide kla-TAT and the cationic anticancer peptide HPRP-A1

In this study, a peptide–peptide co-administration therapy between hybrid peptide kla-TAT and cationic anticancer peptide HPRP-A1 was designed to increase the anticancer activity of the combination peptides through synergistic effect. kla is a pro-apoptotic peptide which could induce rapid cancer cell apoptosis by disruption the mitochondrial membrane when internalized the cells. To enhance more kla peptides pass through cell membrane, a double improvement strategy was designed by chemically conjugation with cell penetration peptide TAT as well as co-administration with cationic membrane active peptide HPRP-A1, and the double anticancer mechanism of the kla-TAT peptide and HPRP-A1 including membrane disruption and apoptosis induction was verified through in vitro experiments. The CompuSyn synergism/antagonism analysis showed that kla-TAT acted synergistically with HPRP-A1 against a non-small cell lung cancer (NSCLC) A549 cell line. The anticancer activities of the two peptides were dramatically increased by co-administration, under the mechanism of cell membrane disruption, caspase-dependent apoptosis induction, as well as cyclin-D1 down-regulation based G1 phase arrest. We believe that the synergic therapeutic strategy would be a meaningful method for the anticancer peptides used in cancer treatment.     

Buforins: Histone H2A-derived antimicrobial peptides from toad stomach

Antimicrobial peptides (AMPs) constitute an important component of the innate immune system in a variety of organisms. Buforin I is a 39-amino acid AMP that was first isolated from the stomach tissue of the Asian toad Bufo bufo gargarizans. Buforin II is a 21-amino acid peptide that is derived from buforin I and displays an even more potent antimicrobial activity than its parent AMP. Both peptides share complete sequence identity with the N-terminal region of histone H2A that interacts directly with nucleic acids. Buforin I is generated from histone H2A by pepsin-directed proteolysis in the cytoplasm of gastric gland cells. After secretion into the gastric lumen, buforin I remains adhered to the mucous biofilm that lines the stomach, thus providing a protective antimicrobial coat. Buforins, which house a helix-hinge-helix domain, kill a microorganism by entering the cell without membrane permeabilization and thus binding to nucleic acids. The proline hinge is crucial for the cell penetrating activity of buforins. Buforins also are known to possess anti-endotoxin and anticancer activities, thus making these peptides attractive reagents for pharmaceutical applications. This review describes the role of buforins in innate host defense; future research paradigms; and use of these agents as human therapeutics.     

Peptide ligand‐mediated endocytosis of nanoparticles to cancer cells: Cell receptor‐binding‐ versus cell membrane‐penetrating peptides

The endocytosis‐mediating performances of two types of peptide ligands, cell receptor binding peptide (CRBP) and cell membrane penetrating peptide (CMPP), were analyzed and compared using a common carrier of peptide ligands‐human ferritin heavy chain (hFTH) nanoparticle. Twenty‐four copies of a CMPP(human immunodeficiency virus‐derived TAT peptide) and/or a CRBP (peptide ligand with strong and specific affinity for either human integrin(α_vβ₃) or epidermal growth factor receptor I (EGFR) that is overexpressed on various cancer cells) were genetically presented on the surface of each hFTH nanopariticle. The quantitative level of endocytosis and intracellular localization of fluorescence dye‐labeled CRBP‐ and CMPP‐presenting nanoparticles were estimated in the in vitro cultures of integrin‐ and EGFR‐overexpressing cancer and human dermal fibroblast cells(control). From the cancer cell cultures treated with the CMPP‐ and CRBP‐presenting nanoparticles, it was notable that CRBPs resulted in quantitatively higher level of endocytosis than CMPP (TAT) and successfully transported the nanoparticles to the cytosol of cancer cells depending on concentration and treatment period of time, whereas TAT‐mediated endocytosis localized most of the nanoparticles within endosomal vesicles under the same conditions. These novel findings provide highly useful informations to many researchers both in academia and in industry who are interested in developing anticancer drug delivery systems/carriers.     

Studies on anticancer activities of antimicrobial peptides

In spite of great advances in cancer therapy, there is considerable current interest in developing anticancer agents with a new mode of action because of the development of resistance by cancer cells towards current anticancer drugs. A growing number of studies have shown that some of the cationic antimicrobial peptides (AMPs), which are toxic to bacteria but not to normal mammalian cells, exhibit a broad spectrum of cytotoxic activity against cancer cells. Such studies have considerably enhanced the significance of AMPs, both synthetic and from natural sources, which have been of importance both for an increased understanding of the immune system and for their potential as clinical antibiotics. The electrostatic attraction between the negatively charged components of bacterial and cancer cells and the positively charged AMPs is believed to play a major role in the strong binding and selective disruption of bacterial and cancer cell membranes, respectively. However, it is unclear why some host defense peptides are able to kill cancer cells when others do not. In addition, it is not clear whether the molecular mechanism(s) underlying the antibacterial and anticancer activities of AMPs are the same or different. In this article, we review various studies on different AMPs that exhibit cytotoxic activity against cancer cells. The suitability of cancer cell-targeting AMPs as cancer therapeutics is also discussed.     

New lytic peptides based on the D,L-amphipathic helix motif preferentially kill tumor cells compared to normal cells

Despite significant advances in cancer therapy, there is an urgent need for drugs with a new mode of action that will preferentially kill cancer cells. Several cationic antimicrobial peptides, which bind strongly to negatively charged membranes, were shown to kill cancer cells slightly better than normal cells. This was explained by a slight increase (3-9%) in the level of the negatively charged membrane phosphatidylserine (PS) in many cancer cells compared to their normal counterparts. Unfortunately, however, these peptides are inactivated by serum components. Here we synthesized and investigated the anticancer activity and the role of peptide charge, peptide structure, and phospholipid headgroup charge on the activity of a new group of diastereomeric lytic peptides (containing D- and L-forms of leucine and lysine; 15-17 amino acids long). The peptides are highly toxic to cancer cells, to a degree similar to or larger than that of mitomycin C. However, compared with mitomycin C and many native antimicrobial peptides, they are more selective for cancer cells. The peptides were investigated for (i) their binding to mono- and bilayer membranes by using the surface plasmon resonance (SPR) technique, (ii) their ability to permeate membranes by using fluorescence spectroscopy, (iii) their structure and their effect on the lipid order by using ATR-FTIR spectroscopy, and (iv) their ability to bind to cancer versus normal cells by using confocal microscopy. The data suggest that the peptides disintegrate the cell membrane in a detergent-like manner. However, in contrast to native antimicrobial peptides, the diastereomers bind and permeate similarly zwitterionic and PS-containing model membranes. Therefore, cell selectivity is probably determined mainly by improved electrostatic attraction of the peptides to acidic components on the surface of cancer cells (e.g., O-glycosylation of mucines). The simple composition of the diastereomeric peptides and their stability regarding enzymatic degradation by serum components make them excellent candidates for new chemotherapeutic drugs.     

TAT Modification of Alpha-Helical Anticancer Peptides to Improve Specificity and Efficacy

HPRP-A1 is an amphipathic α-helical anticancer peptide (ACP) derived from the N-terminus of ribosomal protein L1 (RpL1) of Helicobacter pylori. In our previously study, HPRP-A1 has been reported that induced HeLa cell apoptosis in a caspase-dependent approach and involved both by the death receptor ‘extrinsic’ pathway and the mitochondria ‘intrinsic’ pathway. Here we report the construction of a new hybrid peptide, HPRP-A1-TAT, comprising the cell-permeating peptide TAT linked to the C-terminus of HPRP-A1. This peptide exhibits higher anticancer activity against HeLa cells with lower toxicity against human RBC than HPRP-A1. Two FITC-labeled peptides, FITC-HPRP-A1 and FITC-HPRP-A1-TAT, were used to investigate and compare the cellular uptake mechanism using fluorescence spectra and flow cytometry. Compared with HPRP-A1, HPRP-A1-TAT quickly crossed cell, entered the cytoplasm via endocytosis, and disrupted the cell membrane integrity. HPRP-A1-TAT exhibited stronger anticancer activity than HPRP-A1 at the same concentration by increasing early apoptosis of HeLa cells and inducing caspase activity. Notably, after 24 h, the cellular concentration of HPRP-A1-TAT was higher than that of HPRP-A1. This result suggests that TAT protects HPRP-A1 against degradation, likely due to its high number of positively charged amino acids or the further release of peptides into cancer cells from endocytotic vesicles. We believe that this TAT modification approach may provide an effective new strategy for improving the therapeutic index and anticancer activity of ACPs for clinical use.     

The effect of a beta‐lactamase inhibitor peptide on bacterial membrane structure and integrity: a comparative study

Co‐administration of beta‐lactam antibiotics and beta‐lactamase inhibitors has been a favored treatment strategy against beta‐lactamase‐mediated bacterial antibiotic resistance, but the emergence of beta‐lactamases resistant to current inhibitors necessitates the discovery of novel non‐beta‐lactam inhibitors. Peptides derived from the Ala46–Tyr51 region of the beta‐lactamase inhibitor protein are considered as potent inhibitors of beta‐lactamase; unfortunately, peptide delivery into the cell limits their potential. The properties of cell‐penetrating peptides could guide the design of beta‐lactamase inhibitory peptides. Here, our goal is to modify the peptide with the sequence RRGHYY that possesses beta‐lactamase inhibitory activity under in vitro conditions. Inspired by the work on the cell‐penetrating peptide pVEC, our approach involved the addition of the N‐terminal hydrophobic residues, LLIIL, from pVEC to the inhibitor peptide to build a chimera. These residues have been reported to be critical in the uptake of pVEC. We tested the potential of RRGHYY and its chimeric derivative as a beta‐lactamase inhibitory peptide on Escherichia coli cells and compared the results with the action of the antimicrobial peptide melittin, the beta‐lactam antibiotic ampicillin, and the beta‐lactamase inhibitor potassium clavulanate to get mechanistic details on their action. Our results show that the addition of LLIIL to the N‐terminus of the beta‐lactamase inhibitory peptide RRGHYY increases its membrane permeabilizing potential. Interestingly, the addition of this short stretch of hydrophobic residues also modified the inhibitory peptide such that it acquired antimicrobial property. We propose that addition of the hydrophobic LLIIL residues to the peptide N‐terminus offers a promising strategy to design novel antimicrobial peptides in the battle against antibiotic resistance. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Oligomers of the arginine-rich motif of the HIV-1 TAT protein are capable of transferring plasmid DNA into cells

We constructed multimers of the TAT-(47-57) peptide. This polycationic peptide is known to be a protein and particle transduction domain and at the same time to comprise a nuclear localization function. Here we show that oligomers of the TAT-(47-57) peptide compact plasmid DNA to nanometric particles and stabilize DNA toward nuclease degradation. At optimized vector compositions, these peptides mediated gene delivery to cells in culture 6-8-fold more efficiently than poly-L-arginine or the mutant TAT(2)-M1. When DNA was precompacted with TAT peptides and polyethyleneimine (PEI), Superfect, or LipofectAMINE was added, transfection efficiency was enhanced up to 390-fold compared with the standard vectors. As early as after 4 h of transfection, reporter gene expression mediated by TAT-containing complexes was higher than the 24-h transfection level achieved with a standard PEI transfection. When cells were cell cycle-arrested by serum starvation or aphidicolin, TAT-mediated transfection was 3-fold more efficient than a standard PEI transfection in proliferating cells. In primary nasal epithelial cells and upon intratracheal instillation in vivo, TAT-containing complexes were superior to standard PEI vectors. These data together with confocal imaging of TAT-DNA complexes in cells support the hypothesis that the TAT nuclear localization sequence function is involved in enhancing gene transfer.     

Membrane-active host defense peptides--challenges and perspectives for the development of novel anticancer drugs

Although much progress has been achieved in the development of cancer therapies in recent decades, problems continue to arise particularly with respect to chemotherapy due to resistance to and low specificity of currently available drugs. Host defense peptides as effector molecules of innate immunity represent a novel strategy for the development of alternative anticancer drug molecules. These cationic amphipathic peptides are able to discriminate between neoplastic and non-neoplastic cells interacting specifically with negatively charged membrane components such as phosphatidylserine (PS), sialic acid or heparan sulfate, which differ between cancer and non-cancer cells. Furthermore, an increased number of microvilli has been found on cancer cells leading to an increase in cell surface area, which may in turn enhance their susceptibility to anticancer peptides. Thus, part of this review will be devoted to the differences in membrane composition of non-cancer and cancer cells with a focus on the exposure of PS on the outer membrane. Normally, surface exposed PS triggers apoptosis, which can however be circumvented by cancer cells by various means.Host defense peptides, which selectively target differences between cancer and non-cancer cell membranes, have excellent tumor tissue penetration and can thus reach the site of both primary tumor and distant metastasis. Since these molecules kill their target cells rapidly and mainly by perturbing the integrity of the plasma membrane, resistance is less likely to occur. Hence, a chapter will also describe studies related to the molecular mechanisms of membrane damage as well as alternative non-membrane related mechanisms. In vivo studies have demonstrated that host defense peptides display anticancer activity against a number of cancers such as e.g. leukemia, prostate, ascite and ovarian tumors, yet so far none of these peptides has made it on the market. Nevertheless, optimization of host defense peptides using various strategies to enhance further selectivity and serum stability is expected to yield novel anticancer drugs with improved properties in respect of cancer cell toxicity as well as reduced development of drug resistance.     

Anticancer Peptide SVS-1: Efficacy Precedes Membrane Neutralization

Anticancer peptides are polycationic amphiphiles capable of preferentially killing a wide spectrum of cancer cells relative to noncancerous cells. Their primary mode of action is an interaction with the cell membrane and subsequent activation of lytic effects; however, the exact mechanism responsible for this mode of action remains controversial. Using zeta potential analyses we demonstrate the interaction of a small anticancer peptide with membrane model systems and cancer cells. Electrostatic interactions have a pivotal role in the cell killing process, and in contrast to the antimicrobial peptides action cell death occurs without achieving full neutralization of the membrane charge.     

Designed synthetic analogs of the α‐helical peptide temporin‐La with improved antitumor efficacies via charge modification and incorporation of the integrin αvβ3 homing domain

How to target cancer cells with high specificity and kill cancer cells with high efficiency remains an urgent demand for anticancer drugs. Temporin‐La, which belongs to the family of temporins, presents antitumor activity against many cancer cell lines. We first used a whole bioinformatic analysis method as a platform to identify new anticancer antimicrobial peptides (AMPs). On the basis of these results, we designed a temporin‐La analog (temporin‐Las) and related constructs containing the Arg‐Gly‐Asp (RGD) tripeptide, the integrin αvβ3 homing domain (RGD‐La and RGD‐Las). We detected a link between the net charges and integrin αvβ3 expression of cancer cell lines and the antitumor activities of these peptides. Temporin‐La and its synthetic analogs inhibited cancer cell proliferation in a dose‐dependent manner. Evidence was provided that the affinity between RGD‐Las and tumor cell membranes was stronger than other tested peptides using a pull‐down assay. Morphological changes on the cell membrane induced by temporin‐La and RDG‐Las, respectively, were examined by scanning electron microscopy. Additionally, time‐dependent morphological changes were detected by confocal microscopy, where the binding process of RGD‐Las to the cell membrane could be monitored. The results indicate that the electrostatic interaction between these cationic peptides and the anionic cell membrane is a major determinant of selective cell killing. Thus, the RGD tripeptide is a valuable ligand motif for tumor targeting, which leads to an increased anticancer efficiency by RGD‐Las. These AMP‐derived peptides have clinical potential as specifically targeting agents for the treatment of αvβ3 positive tumors. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Competition between Sec- and TAT-dependent protein translocation in Escherichia coli.

Recently, a new protein translocation pathway, the twin-arginine translocation (TAT) pathway, has been identified in both bacteria and chloroplasts. To study the possible competition between the TAT- and the well-characterized Sec translocon-dependent pathways in Escherichia coli, we have fused the TorA TAT-targeting signal peptide to the Sec-dependent inner membrane protein leader peptidase (Lep). We find that the soluble, periplasmic P2 domain from Lep is re-routed by the TorA signal peptide into the TAT pathway. In contrast, the full-length TorA-Lep fusion protein is not re-routed into the TAT pathway, suggesting that Sec-targeting signals in Lep can override TAT-targeting information in the TorA signal peptide. We also show that the TorA signal peptide can be converted into a Sec-targeting signal peptide by increasing the hydrophobicity of its h-region. Thus, beyond the twin-arginine motif, the overall hydrophobicity of the signal peptide plays an important role in TAT versus Sec targeting. This is consistent with statistical data showing that TAT-targeting signal peptides in general have less hydrophobic h-regions than Sec-targeting signal peptides.     

Antitumor effects and cell selectivity of temporin-1CEa, an antimicrobial peptide from the skin secretions of the Chinese brown frog (Rana chensinensis)

Many antimicrobial peptides from amphibian exhibit additional anticancer properties due to a similar mechanism of action at both bacterial and cancer cells. We have previously reported the cDNA sequence of the antimicrobial peptide temporin-1CEa precursor cloned from the Chinese brown frog Rana chensinensis. In this study, we purified, synthesized and structurally characterized temporin-1CEa from the skin secretions of R. chensinensis. The cytotoxicity and cell selectivity of temporin-1CEa were further examined on twelve human carcinoma cell lines and on normal human umbilical vein smooth muscle cells (HUVSMCs). Our results indicated that temporin-1CEa has the amino acid sequence of FVDLKKIANIINSIF-NH₂, and exhibits 50–56% identity with temporin family peptides from other frog species. The CD spectra for temporin-1CEa adopted a well-defined α-helical structure in 50% TFE/water solution. The results of MTT assay showed that temporin-1CEa exhibits cytotoxicity to all tested cancer cell lines in a concentration-dependent manner, being MCF-7 cells the most sensitive. Moreover, temporin-1CEa had lower hemolytic effect to human erythrocytes and had no significant cytotoxicity to normal HUVSMCs at concentrations showed potent antitumor activity. In summary, temporin-1CEa, an amphiphilic α-helical cationic peptide, may represent a novel anticancer agent for breast cancer therapy, considering its cancer cell selectivity and relatively lower cytotoxicity to normal cells.     

Anticancer activity of CopA3 dimer peptide in human gastric cancer cells

CopA3 is a homodimeric α-helical peptide derived from coprisin which is a defensin-like antimicrobial peptide that was identified from the dung beetle, Copris tripartitus. CopA3 has been reported to have anticancer activity against leukemia cancer cells. In the present study, we investigated the anticancer activity of CopA3 in human gastric cancer cells. CopA3 reduced cell viability and it was cytotoxic to gastric cancer cells in the MTS and LDH release assay, respectively. CopA3 was shown to induce necrotic cell death of the gastric cancer cells by flow cytometric analysis and acridine orange/ethidium bromide staining. CopA3-induced cell death was mediated by specific interactions with phosphatidylserine, a membrane component of cancer cells. Taken together, these data indicated that CopA3 mainly caused necrosis of gastric cancer cells, probably through interactions with phosphatidylserine, which suggests the potential utility of CopA3 as a cancer therapeutic. [BMB Reports 2015; 48(6): 324-329]     

Cationic Antimicrobial Peptides Derived from <Emphasis Type="Italic">Crocodylus siamensis</Emphasis> Leukocyte Extract,Revealing Anticancer Activity and Apoptotic Induction on Human Cervical Cancer Cells

Known antimicrobial peptides KT2 and RT2 as well as the novel RP9 derived from the leukocyte extract of the freshwater crocodile (Crocodylus siamensis) were used to evaluate the ability in killing human cervical cancer cells. RP9 in the extract was purified by a combination of anion exchange column and reversed-phase HPLC, and its sequence was analyzed by mass spectrometry. The novel peptide could inhibit Gram-negative Vibrio cholerae (clinical isolation) and Gram-positive Bacillus pumilus TISTR 905, and its MIC values were 61.2 µM. From scanning electron microscopy, the peptide was seen to affect bacterial surfaces directly. KT2 and RT2, which are designed antimicrobial peptides using the C. siamensis Leucrocin I template, as well as RP9 were chemically synthesized for investigation of anticancer activity. By Sulforhodamine B colorimetric assay, these antimicrobial peptides could inhibit both HeLa and CaSki cancer cell lines. The IC₅₀ values of KT2 and RT2 for HeLa and CaSki cells showed 28.7–53.4 and 17.3–30.8 µM, while those of RP9 were 126.2 and 168.3 µM, respectively. Additionally, the best candidate peptides KT2 and RT2 were used to determine the apoptotic induction on cancer cells by human apoptosis array assay. As a result, KT2 and RT2 were observed to induce apoptotic cell death in HeLa cells. Therefore, these results indicate that KT2 and RT2 with antimicrobial activity have a highly potent ability to kill human cervical cancer cells.     

Anticancer peptide NK-2 targets cell surface sulphated glycans rather than sialic acids

Abstract Some antimicrobial peptides have emerged as potential anticancer agents. In contrast to chemotherapeutics, they act primarily by physical disruption of the cancer cell membrane. Selective targeting of these cationic peptides still remains elusive. We focus on the interaction of α-helical peptides NK-2, cathelicidin LL32, and melittin with PC-3 prostate cancer cells, and we provide strong evidence that, amongst the anionic glycans covering the cell surface, sulphated carbohydrates rather than sialic acids are the preferred interaction sites of the peptides. To test the significance of cell surface carbohydrates, a glycan microarray screen with fluorescently labelled peptides has been performed. Amongst 465 mammalian glycan structures on the chip, more than 20 different sulphated glycans were detected as the preferred binding partners of the peptide NK-2. The amount of peptide bound to sialic acid containing oligosaccharides was close to background level. These findings were consistent with microcalorimetric experiments revealing high and low binding enthalpies of peptides to sulphated carbohydrates and to sialic acid, respectively. Enzymatic desialylation of PC-3 cells did not affect peptide-mediated changes in cell metabolism, cell membrane permeabilisation, killing rate, and kinetics. Finally, the cytotoxicity of all peptides could be drastically impaired through the competitive inhibition by chondroitin sulphate, but not by sialic acid and sialylated fetuin.     

Measuring peptide translocation into large unilamellar vesicles

There is an active interest in peptides that readily cross cell membranes without the assistance of cell membrane receptors(1). Many of these are referred to as cell-penetrating peptides, which are frequently noted for their potential as drug delivery vectors(1-3). Moreover, there is increasing interest in antimicrobial peptides that operate via non-membrane lytic mechanisms(4,5), particularly those that cross bacterial membranes without causing cell lysis and kill cells by interfering with intracellular processes(6,7). In fact, authors have increasingly pointed out the relationship between cell-penetrating and antimicrobial peptides(1,8). A firm understanding of the process of membrane translocation and the relationship between peptide structure and its ability to translocate requires effective, reproducible assays for translocation. Several groups have proposed methods to measure translocation into large unilamellar lipid vesicles (LUVs)(9-13). LUVs serve as useful models for bacterial and eukaryotic cell membranes and are frequently used in peptide fluorescent studies(14,15). Here, we describe our application of the method first developed by Matsuzaki and co-workers to consider antimicrobial peptides, such as magainin and buforin II(16,17). In addition to providing our protocol for this method, we also present a straightforward approach to data analysis that quantifies translocation ability using this assay. The advantages of this translocation assay compared to others are that it has the potential to provide information about the rate of membrane translocation and does not require the addition of a fluorescent label, which can alter peptide properties(18), to tryptophan-containing peptides. Briefly, translocation ability into lipid vesicles is measured as a function of the Foster Resonance Energy Transfer (FRET) between native tryptophan residues and dansyl phosphatidylethanolamine when proteins are associated with the external LUV membrane (Figure 1). Cell-penetrating peptides are cleaved as they encounter uninhibited trypsin encapsulated with the LUVs, leading to disassociation from the LUV membrane and a drop in FRET signal. The drop in FRET signal observed for a translocating peptide is significantly greater than that observed for the same peptide when the LUVs contain both trypsin and trypsin inhibitor, or when a peptide that does not spontaneously cross lipid membranes is exposed to trypsin-containing LUVs. This change in fluorescence provides a direct quantification of peptide translocation over time.     

一种猪溶菌酶来源的抗菌六肽的分离鉴定及其性质

为提高猪溶菌酶(Sus scrofa lysozyme,SSL)的抗革兰氏阴性菌活性,将其进行了不同蛋白酶的水解,选择抗革兰氏阴性菌效果最好的水解产物,利用凝胶过滤色谱和反相制备色谱进行分离,对其功能成分进行液质联用鉴定。对分离得到的物质进行抗菌活性验证和生物信息学的分析,并在此基础上对抗菌物质的杀菌机理进行了探讨。结果表明,胰蛋白酶的水解产物具有较高的杀灭革兰氏阴性菌的活性,进一步分离纯化得到了具有抗革兰氏阴性菌活性的六肽A-W-V-A-W-K。经化学合成验证,该六肽既保留了SSL的部分抗菌活性,也具备杀灭多种革兰氏阴性菌的能力。进一步分析发现其位于SSL分子C端的一个螺旋-回环-螺旋的结构中,并由此推测其杀菌机理是通过改变细胞膜的渗透性,进而使细胞内溶物流出而造成细胞死亡,而抗菌实验也验证了这一推测。该抗菌肽的发现为后续提高SSL的抗菌活性提供了理论依据。