Effects of the hinge region of cecropin A(1-8)-magainin 2(1-12), a synthetic antimicrobial peptide, on liposomes, bacterial and tumor cells

A 20-residue hybrid peptide (CA(1-8)-MA(1-12): KWKLFKKIGIGKFLHSAKKF-NH(2)) incorporating 1-8 residues of cecropin A (CA) and 1-12 residues of magainin 2 (MA) has potent antibiotic activity without hemolytic activity. In order to investigate the effects of the flexible hinge sequence, Gly-Ile-Gly of CA(1-8)-MA(1-12) (CA-MA) on antibiotic activity, CA-MA and its three analogues, CA-MA1, CA-MA2 and CA-MA3 were synthesized. The Gly-Ile-Gly sequence of CA-MA was deleted in CA-MA1 and replaced with Pro and Gly-Pro-Gly in CA-MA2 and CA-MA3, respectively. CA-MA1 and CA-MA3 caused a significant decrease in the bactericidal rate against Escherichia coli and Bacillus subtilis and the tumoricidal activity against four different tumor cells, and the PC/PS (4:1, w/w) vesicle-aggregating and disrupting activities. However, CA-MA2 showed a similar bactericidal rate and antitumor, vesicle-aggregating and disrupting activities, as compared with CA-MA. These results suggested that the flexibility or beta-turn induced by Gly-Ile-Gly or Pro in the central part of CA-MA may be important in the electrostatic interaction of the cationic short alpha-helical region in the N-terminus with the cell membrane surface and the hydrophobic interaction of amphipathic alpha-helical region in the C-terminus with the hydrophobic acyl chains in the cell membrane. CA-MA3 exhibited lower activity in antibacterial, antitumor, and vesicle-aggregating and disrupting activities than CA-MA and CA-MA2. This result suggested that the excessive beta-turn structure by Gly-Pro-Gly in CA-MA3 seems to interrupt the ion channel/pore formation on the lipid bilayer. It was concluded that the appropriate flexibility or beta-turn structure provided by the central hinge is responsible for the effective antibiotic activity of the antimicrobial peptides with the helix-hinge-helix structure.     

Role of the hinge region and the tryptophan residue in the synthetic antimicrobial peptides, cecropin A(1-8)-magainin 2(1-12) and its analogues, on their antibiotic activities and structures

A 20-residue hybrid peptide CA(1-8)-MA(1-12) (CA-MA), incorporating residues 1-8 of cecropin A (CA) and residues 1-12 of magainin 2 (MA), has potent antimicrobial activity without toxicity against human erythrocytes. To investigate the effects of the Gly-Ile-Gly hinge sequence of CA-MA on the antibacterial and antitumor activities, two analogues in which the Gly-Ile-Gly sequence of CA-MA is either deleted (P1) or substituted with Pro (P2) were synthesized. The role of the tryptophan residue at position 2 of CA-MA on its antibiotic activity was also investigated using two analogues, in which the Trp2 residue of CA-MA is replaced with either Ala (P3) or Leu (P4). The tertiary structures of CA-MA, P2, and P4 in DPC micelles, as determined by NMR spectroscopy, have a short amphiphilic helix in the N-terminus and about three turns of alpha-helix in the C-terminus, with the flexible hinge region between them. The P1 analogue has an alpha-helix from Leu4 to Ala14 without any hinge structure. P1 has significantly decreased lytic activities against bacterial and tumor cells and PC/PS vesicles (3:1, w/w), and reduced pore-forming activity on lipid bilayers, while P2 retained effective lytic activities and pore-forming activity. The N-terminal region of P3 has a flexible structure without any specific secondary structure. The P3 modification caused a drastic decrease in the antibiotic activities, whereas P4, with the hydrophobic Leu side chain at position 2, retained its activities. On the basis of the tertiary structures, antibiotic activities, vesicle-disrupting activities, and pore-forming activities, the structure-function relationships can be summarized as follows. The partial insertion of the Trp2 of CA-MA into the membrane, as well as the electrostatic interactions between the positively charged Lys residues at the N-terminus of the CA-MA and the anionic phospholipid headgroups, leads to the primary binding to the cell membrane. Then, the flexibility or bending potential induced by the Gly-Ile-Gly hinge sequence or the Pro residue in the central part of the peptides may allow the alpha-helix in the C-terminus to span the lipid bilayer. These structural features are crucial for the potent antibiotic activities of CA-MA.     

NMR structural characterization of cecropin A(1-8) - magainin 2(1-12) and cecropin A (1-8) - melittin (1-12) hybrid peptides.

In order to elucidate the structure-antibiotic activity relationships of the peptides, the three-dimensional structures of two hybrid peptides, CA(1-8) - MA(1-12) and CA(1-8) - ME(1-12) in trifluoroethanol-containing aqueous solution were investigated by NMR spectroscopy. Both CA(1-8) - MA(1-12) and CA(1-8) - ME(1-12) have strong antibacterial activity but only CA(1-8) - ME(1-12) has hemolytic activity against human erythrocytes. CA(1-8) - MA(1-12) has a hydrophobic 310-helix of only two turns combined with one short helix in the N-terminus with a flexible hinge section in between. CA(1-8) - MA(1-12) has a severely bent structure in the middle of the peptide. These structural features as well as the low hydrophobicity of CA(1-8) - MA(1-12) seem to be crucial for the selective lysis against the membrane of prokaryotic cells. CA(1-8) - ME(1-12) has an alpha-helical structure of about three turns in the melittin domain and a flexible structure with one turn in the cecropin domain connected with a flexible hinge section in between, and these might be the structural features required for membrane disruption against prokaryotic and eukaryotic cells. The central hinge region (Gly9-Ile10-Gly11) in an amphipathic antibacterial peptide is considered to play an important role in providing the conformational flexibility required for ion channel formation of the C-terminal hydrophobic alpha-helix on cell membrane.     

Structure-antibacterial activity relationship of cecropin A derivatives

To investigate the effect of net positive charge, alpha-helicity and hydrophobicity of the peptides on antibacterial activity, we designed three kinds of cecropin A (CA) derivatives. Compared to CA, F3 with the highest net positive charge exhibited similar or slightly weaker antibacterial activity (MIC: 3.13 approximately 6.25 microM). F1 showed lower antibacterial activity than that of F3, even though it has higher hydrophobicity and a-helicity than F3. This result indicates that the net positive charge of cecropin A-like peptides appears to be a more important factor in antibacterial activity than alpha-helicity and hydrophobicity. The two peptides F1 and F2 possessed almost similar antibacterial activity, but F2 showed very lower activity in the membrane disruption than F1, suggesting other factors are involved in the antibacterial activity of the peptides as well as peptide-lipid interaction.     

Structure and fungicidal activity of a synthetic antimicrobial peptide, P18, and its truncated peptides

P18 (KWKLFKKIPKFLHLAKKF-NH2) is an antimicrobial peptide designed from a cecropin A-magainin 2 hybrid that has potent antibacterial activity without hemolytic activity against human erythrocytes. In this study, P18 displayed potent fungicidal activity (MIC: 12.5 approximately 25 microM) against pathogenic fungi, Candida albicans, Trichosporon beigelii, Aspergillus flavus and Fusarium oxyspovrum. The central Pro9 residue and the entire sequence of P18 are essential for its full fungicidal activity. Circular dichroism analysis suggested that the higher alpha-helical content of the peptides did not correlate with the stronger fungicidal activity.     

Antibacterial, antitumor and hemolytic activities of alpha-helical antibiotic peptide, P18 and its analogs.

The alpha-helical antibiotic peptide (P18: KWKLFKKIPKFLHLAKKF-NH2) designed from the cecropin A(1-8)-magainin 2 (1-12) hybrid displayed strong bactericidal and tumoricidal activity without inducing hemolysis. The effect of the Pro9 residue at central position of P18 on cell selectivity was investigated by Pro9 --> Leu or Pro9 --> Ser substitution. Either substitution markedly reduced the antibacterial activity of P18 and increased hemolysis, although it did not significantly affect cytotoxicity against human transformed tumor and normal fibroblast cells. These results suggest that a proline kink in alpha-helical antibiotic peptide P18 serves as a hinge region to facilitate ion channel formation on bacterial cell membranes and thus plays an important role in providing high selectivity against bacterial cells. Furthermore, to investigate the structure-antibiotic activity relationships of P18, a series of N- or C-terminal deletion and substitution analogs of P18 were synthesized. The C-terminal region of P18 was related to its antibiotic activity and alpha-helical conformation on lipid membranes rather than N-terminal one. Higher alpha-helicity of the peptides was involved in the hemolytic and antitumor activity rather than antibacterial activity. Except for [L9]-P18 and [S9]-P18, all the designed peptides containing a Pro residue showed potent antibacterial activity, although they did not induce a cytolytic effect against human erythrocyte and normal fibroblast cells at the concentration required to kill bacteria. In particular, P18 and some analogs (N-1, N-2, N-3, N-3L and N-4L) with potent bactericidal and tumoricidal activity and little or no normal cell toxicity may serve as an attractive candidate for the development of novel anti-infective or antitumor agents.     

Synergism of Leu-Lys rich antimicrobial peptides and chloramphenicol against bacterial cells

To investigate the antibiotic activity and synergistic effect, analogues were designed to increase not only net positive charge by Lys-substitution but also hydrophobic helix region by Leu-substitution from CA (1-8)-MA (1-12) hybrid peptide (CA-MA). In particular, CA-MA analogue P5 (P5), designed by flexible region (GIG-->P)-substitution, Lys- (positions 4, 8, 14, 15) and Leu- (positions 5, 6, 12, 13, 16, 17, 20) substitutions, showed potent antibacterial activity in minimal inhibition concentration (MIC) and minimal bactericidal concentration (MBC) without having hemolytic activity. In addition, P5 and chloramphenicol has potent synergistic effect against tested cell lines. As determined by propidium iodide (PI) staining, flow cytometry showed that P5 plus chloramphenicol-treated cells had higher fluorescence intensity than untreated, P5- and chloramphenicol-treated cells. The effect on plasma membrane was examined by investigating the transmembrane potential depolarizing experiments of S. aureus with P5 and chloramphenicol. The result showed that the peptide exerts its antibacterial activity by acting on the plasma membrane. Furthermore, P5 caused significant morphological alterations of S. aureus, as shown by scanning electron microscopy. Our results suggest that peptide P5 is an excellent candidate as a lead compound for the development of novel anti-infective agents and synergistic effects with conventional antibiotic agents but lack hemolytic activity.     

Conformational study of a custom antibacterial peptide cecropin B1: implications of the lytic activity

Cecropin B1 (CB1) with two amphipathic alpha-helical segments is a derivative of the natural antibacterial peptide, cecropin B. The assays of cell lysis show that, compared with cecropin A (CA), CB1 has a similar ability to lyse bacteria with a higher potency (two- to six-fold higher) in killing cancer cells. The difference may be due to the fact that the peptides possess different structures and sequences. In this study, the solution structure of CB1 in 20% hexafluoroisopropanol was determined by two-dimensional nuclear magnetic resonance (NMR) spectroscopy. The (1)H NMR resonances were assigned. A total of 350 inter-proton distances were used to calculate the solution structure of CB1. The final ensemble structures were well converged, showing the minimum root mean square deviation. The results indicate that CB1 has two stretches of helices spanning from residues 3 to 22 and from residues 26 to 33, which are connected by a hinge section formed by Gly-23 and Pro-24. Lys-25 is partially incorporated in the hinge region. The bent angle between two helical segments located in two planes was between 100 and 110 degrees. With comparisons of the known NMR structure of CA and its activities on bacteria and cancer cells, the structure-function relationship of the peptides is discussed.     

Comparative activities of cecropin A, melittin, and cecropin A-melittin peptide CA(1-7)M(2-9)NH2 against multidrug-resistant nosocomial isolates of Acinetobacter baumannii

The in vitro activity of three polycationic peptides, cecropin A, melittin, and cecropin A-melittin hybrid peptide CA(1-7)M(2-9)NH2, alone and in combination with various clinically used antimicrobial agents, was investigated against 32 nosocomial isolates of Acinetobacter baumannii. Antimicrobial activities were measured by MIC, MBC and bacterial killing assay. The peptides demonstrated different ranges of inhibitory values: overall, the organisms were more susceptible to CA(1-7)M(2-9)NH2 (MIC range, 0.25-16 mg/l) than to cecropin A (0.50-32 mg/l) and melittin (0.50-32 mg/l). Synergy was observed when CA(1-7)M(2-9)NH2 and melittin were combined with beta-lactam antibiotics.     

A Leu-Lys-rich antimicrobial peptide: activity and mechanism

To develop novel antibiotic peptides useful as therapeutic drugs, the analogues were designed to increase not only net positive charge by Lys substitution but also hydrophobic helix region by Leu substitution from cecropin A (1-8)-magainin 2 (1-12) hybrid peptide (CA-MA). In particular, CA-MA analogue P5 (P5), designed by flexible region (GIG-->P) substitution, Lys (positions 4, 8, 14, 15) and Leu (positions 5, 6, 12, 13, 16, 17, 20) substitutions, showed an enhanced antimicrobial and antitumor activity without hemolysis. Confocal microscopy showed that P5 was located in the plasma membrane. The antibacterial effects of analogues were further confirmed by using 1,6-diphenyl-1,3,5-hexatriene as a plasma membrane probe. Flow cytometric analysis revealed that P5 acted in an energy-independent manner. This interaction is also independent of the ionic environment. Furthermore, P5 causes significant morphological alterations of the bacterial surfaces as shown by scanning electron microscopy and showed strong membrane disrupting activity when examined using liposomes (phosphatidyl choline/cholesterol; 10:1, w/w). Its potent antibiotic activity suggests that P5 is an excellent candidate as a lead compound for the development of novel antiinfective agents.     

A Leu–Lys-rich antimicrobial peptide: activity and mechanism

To develop novel antibiotic peptides useful as therapeutic drugs, the analogues were designed to increase not only net positive charge by Lys substitution but also hydrophobic helix region by Leu substitution from cecropin A (1–8)–magainin 2 (1–12) hybrid peptide (CA–MA). In particular, CA–MA analogue P5 (P5), designed by flexible region (GIG→P) substitution, Lys (positions 4, 8, 14, 15) and Leu (positions 5, 6, 12, 13, 16, 17, 20) substitutions, showed an enhanced antimicrobial and antitumor activity without hemolysis. Confocal microscopy showed that P5 was located in the plasma membrane. The antibacterial effects of analogues were further confirmed by using 1,6-diphenyl-1,3,5-hexatriene as a plasma membrane probe. Flow cytometric analysis revealed that P5 acted in an energy-independent manner. This interaction is also independent of the ionic environment. Furthermore, P5 causes significant morphological alterations of the bacterial surfaces as shown by scanning electron microscopy and showed strong membrane disrupting activity when examined using liposomes (phosphatidyl choline/cholesterol; 10:1, w/w). Its potent antibiotic activity suggests that P5 is an excellent candidate as a lead compound for the development of novel antiinfective agents.     

N-terminal analogues of cecropin A: synthesis, antibacterial activity, and conformational properties

Six analogues of the 37-residue antibacterial peptide cecropin A were synthesized by the solid-phase method: cecropin A-(2-37), [Glu2]cecropin A, [Pro4]cecropin A, [Glu6]cecropin A, [Leu6]cecropin A, and [Pro8]cecropin A. Their antibacterial activities against four test organisms were determined and related to conformational changes observed in their CD spectra and were discussed on the basis of a previously proposed amphipathic alpha-helix model. An aromatic residue in position 2 was shown to be important for activity against all tested bacteria. The highly alpha-helical 1-11 region of cecropin A did not appear to play a significant role in its activity against Escherichia coli but was clearly involved in its interaction against Pseudomonas aeruginosa, Bacillus megaterium, and Micrococcus luteus.     

The chemical synthesis of cecropin D and an analog with enhanced antibacterial activity

Cecropin D was synthesized by solid-phase methods and shown to be homogeneous and of correct composition and molecular weight. It was indistinguishable from natural cecropin D and constitutes a structure proof for this peptide. Several analogs of cecropin D were synthesized and used to draw conclusions about the structural features contributing to antibacterial activity. They included [Lys1]cecropin D, [Gln3, Leu4] cecropin D, and cecropin D-(9-37). It was concluded that a strongly basic NH2-terminal segment is a prerequisite for antibacterial activity. A hybrid analog cecropin A-(1-11) D-(12-37) was designed and predicted to have enhanced potency. It was found to be 5 to 55 times as active as cecropin D against six of the bacteria tested and was slightly more active than cecropin A. However, against Bacillus subtilis Bs11 the analog was 6 times more active than cecropin A.     

Antibacterial and antimalarial properties of peptides that are cecropin-melittin hybrids

Solid phase synthesis was used to produce 5 hybrid peptides containing sequences from the antibacterial peptide, cecropin A, and from the bee venom toxin, melittin. Four of these chimeric peptides showed good antibacterial activity against representative Gram-negative and Gram-positive bacterial species. The best hybrid, cecropin A(1-13)-melittin(1-13) was 100-fold more active than cecropin A against Staphylococcus aureus. It was also a 10-fold better antimalarial agent than cecropin B or magainin 2. Sheep red cells were lysed by melittin at low concentrations, but not by the hybrid molecules, even at 50 times higher concentrations.     

Solution structure and cell selectivity of piscidin 1 and its analogues

Piscidin 1 (Pis-1) is a novel cytotoxic peptide with a cationic alpha-helical structure that was isolated from the mast cells of hybrid striped bass [Silphaduang, U., and Noga, E. J. (2001) Nature 414, 268-269]. Pis-1 is not selective for bacterial versus mammalian cells. In the present study, to develop novel antibiotic peptides with selectivity for bacterial cells, we examined the effect of substituting two glycine residues, Gly8 and Gly13, with Ala or Pro on this peptide's structure and biological activities. The bacterial cell selectivity of the peptides decreased in the following order: Gly-->Pro analogues > Gly-->Pro/Ala analogues > Pis-1 > Gly-->Ala analogues. The antimicrobial and hemolytic activities and abilities to permeabilize the model phospholipid membranes were higher for Pis-1 with Gly or Pro at position 8 than for its counterparts with either Gly or Pro at position 13. We determined the tertiary structure of Pis-1 and its analogues in the presence of SDS micelles by NMR spectroscopy. We found that Pis-1 has an alpha-helical structure from Phe2 to Thr21. Also, Pis-1 AA (Gly8, Gly13-->Ala8, Ala13) with higher antibacterial and hemolytic activity than Pis-1 has a stable alpha-helical structure from Phe2 to Thr21. Pis-1 PG (Gly-->Pro8) with bacterial cell selectivity has a hinge structure at Pro8, which provides flexibility in piscidin, followed by a three-turn helix from Val10 to Gly22 in the C-terminal region. Taken together, our results demonstrate that the conformational flexibility provided by introduction of a Pro at position 8, coupled with the primary anchoring of phenylalanines and histidines in the N-terminus to the cell membrane and the optimal length of the C-terminal amphipathic alpha-helix, are the critical factors that confer antibacterial activity and bacterial cell selectivity to Pis-1 PG. Pis-1 PG may be a good candidate for the development of a new drug with potent antibacterial activity but without cytotoxicity.     

家蚕天蚕素cDNA原核表达及抗菌活性检测

采用RT-PCR方法从家蚕Bombyx mori新疆品种新蚕三号组织中扩增天蚕素cDNA片段,回收并克隆至Pmd18-T载体,进行序列分析。基因序列分析结果与已发表的天蚕素B的序列同源性为98%,表明所克隆的新疆家蚕天蚕素cDNA为独特的cDNA片段。将天蚕素基因与Pgex-4T-1融合表达载体中的谷胱甘肽转移酶基因融合,在大肠杆菌中表达, 结果表明经IPTG 诱导30 min后,pGEX-4T-1/天蚕素转化后的大肠杆菌生长明显受到抑制;当诱导210 min 后,大肠杆菌数量又开始增加,逐渐恢复至正常水平。说明天蚕素与谷胱甘肽转移酶基因融合表达后,在IPTG存在的短时间内仍然对原核细胞有较强的抗菌抑杀作用。     

Hematological and antifungal properties of temporin A and a cecropin A-temporin A hybrid

Temporin A (TA) and a cecropin A-temporin A hybrid peptide (CATA) were synthesized and assayed for their hemolytic, anticoagulant, and antifungal properties. CATA retains significant antifungal activity, is less hemolytic than TA, and inhibits blood coagulation. These results recommend further studies of the biological activities of CATA.     

Design of novel analogues with potent antibiotic activity based on the antimicrobial peptide, HP(2-9)-ME(1-12)

To develop novel antibiotic peptides useful as therapeutic drugs, a number of analogues were designed to increase the hydrophobic helix region either by Trp-substitution or net positive charge increase by Lys-substitution, from HP(2-9)-ME(1-12). The antibiotic activities of these peptides were evaluated using bacterial (Salmonella tryphimurium, Proteus vulgaris, Bacillus subtilis and Staphylococcus aureus), fungi (Saccharomyces cerevisiae, Trichosporon beigelii and Candida albicans), tumor and human erythrocyte cells. The substitution of Lys for Thr at position 18 and 19 of HP(2-9)-ME(1-12) (HM5) increased activity against Proteus vulgaris and fungal strains without hemolysis. In contrast, substitution of Trp for Lys and Thr at positions 2, 15 and 19 of HP(2-9)-ME(1-12), respectively (HM3 and HM4), decreased activity but increased hemolysis against human erythrocytes. This suggests that an increase in positive charge increases antimicrobial activity whereas an increase in hydrophobicity by introducing Trp residues at C-terminus of HP(2-9)-ME(1-12) causes a hemolytic effect. Circular dichroism spectra suggested that the alpha-helical structure of these peptides plays an important role in their antibiotic effect but that the alpha-helical property is not connected with the enhanced antibiotic activity.     

Influence of the N- and C-terminal regions of leu-lys rich antimicrobial peptide on antimicrobial activity

P5 (KWKKLLKKPLLKKLLKKL-NH(2)) is an antibacterial 18-mer Leu-Lys rich peptide from CA (1-8)-MA (1-12) hybrid peptide (CA-MA). Here we show that decreasing the net hydrophobicity and charge of CA-MA by deleting Leu- or Lys- of the N- or C-terminal regions of P5 (P10 or P11). The antimicrobial activity of the peptides was measured by their growth inhibitory effect upon S. aureus, B. subtilis, P. aeruginosa, S. typhimurium, E. coli, T. beigelii and C. albicans. Antimicrobial activity required a full length C-terminus. Confocal microscopy showed that P11 was located in the plasma membrane. In this study, P11, K(3)K(4)L(5)L(6)-deleted peptide, acted independent on the ionic environment. Furthermore, P11 causes significant morphological alterations of the fungal surfaces as shown by scanning electron microscopy.