Induced Resistance to the Designer Proline-rich Antimicrobial Peptide A3-APO does not Involve Changes in the Intracellular Target DnaK

The designer proline-rich antimicrobial peptide A3-APO and its Chex1-Arg20 single chain in vivo metabolite were studied for their ability to induce bacterial resistance upon repeated incubation of Escherichia coli and Klebsiella pneumoniae strains in sublethal concentrations. While no resistant E. coli phenotype emerged to either peptides, after 10 passages the K. pneumoniae strain became resistant to the monomer but not the dimer. The major microbiological difference between A3-APO and Chex1-Arg20 is the improved membrane-disintegrating ability of the dimeric prodrug. Thus, in agreement with earlier studies, the induced resistance likely resides in some membrane component rather than the intracellular target protein DnaK. In support, no genetic alteration in the DnaK multihelical lid region could be observed in any of the sensitive or resistant bacterial strains.     

Antimicrobial peptides in the first line defence of human colon mucosa

Antimicrobial peptides and proteins are effector molecules in the protection of epithelial surfaces. We have evaluated the presence of antimicrobial peptides/proteins that can participate in human colonic defence against microbes. A peptide/protein extract of normal human colon mucosa was found to be active against Gram-positive bacteria, Gram-negative bacteria, and fungi. Four polypeptides with antimicrobial activity were isolated from this material and they were identified by N-terminal amino acid sequence analysis as ubiquicidin, histone H2B, eosinophil cationic protein, and phospholipase A(2) (PLA(2)). Using immunodetection and mass spectrometry, LL-37, HNP1-3, and HBD-1 were also identified. Combined, these results indicate that the colon mucosa is protected by a complex mixture of polypeptides, able to kill invading microbes and working in synergy as a barrier against bacterial invasion.     

Design,structural and functional characterization of a Temporin-1b analog active against Gram-negative bacteria

Background

Temporins are small antimicrobial peptides secreted by the Rana temporaria showing mainly activity against Gram-positive bacteria. However, different members of the temporin family, such as Temporin B, act in synergy also against Gram-negative bacteria. With the aim to develop a peptide with a wide spectrum of antimicrobial activity we designed and analyzed a series of Temporin B analogs.

Methods

Peptides were initially obtained by Ala scanning on Temporin B sequence; antimicrobial activity tests allowed to identify the TB_G6A sequence, which was further optimized by increasing the peptide positive charge (TB_KKG6A). Interactions of this active peptide with the LPS of E. coli were investigated by CD, fluorescence and NMR.

Results

TB_KKG6A is active against Gram-positive and Gram-negative bacteria at low concentrations. The peptide strongly interacts with the LPS of Gram-negative bacteria and folds upon interaction into a kinked helix.

Conclusion

Our results show that it is possible to widen the activity spectrum of an antimicrobial peptide by subtle changes of the primary structure. TB_KKG6A, having a simple composition, a broad spectrum of antimicrobial activity and a very low hemolytic activity, is a promising candidate for the design of novel antimicrobial peptides.

General significance

The activity of antimicrobial peptides is strongly related to the ability of the peptide to interact and break the bacterial membrane. Our studies on TB_KKG6A indicate that efficient interactions with LPS can be achieved when the peptide is not perfectly amphipathic, since this feature seems to help the toroidal pore formation process.     

Engineered salt-insensitive alpha-defensins with end-to-end circularized structures

We designed a retro-isomer and seven circularized "beta-tile" peptide analogs of a typical rabbit alpha-defensin, NP-1. The analogs retained defensin-like architecture after the characteristic end-to-end, Cys(3,31) (C I:C VI), alpha-defensin disulfide bond was replaced by a backbone peptide bond. The retro-isomer of NP-1 was as active as the parent compound, suggesting that overall topology and amphipathicity governed its antimicrobial activity. A beta-tile design with or without a single cross-bracing disulfide bond sufficed for antimicrobial activity, and some of the analogs retained activity against Escherichia coli and Salmonella typhimurium in NaCl concentrations that rendered NP-1 inactive. The new molecules had clustered positive charges resembling those in protegrins and tachyplesins, but were less cytotoxic. Such simplified alpha-defensin analogs minimize problems encountered during the oxidative folding of three-disulfide defensins. In addition, they are readily accessible to a novel thia zip cyclization procedure applicable to large unprotected peptide precursors of 31 amino acids in aqueous solutions. Collectively, these findings provide new and improved methodology to create salt-insensitive defensin-like peptides for application against bacterial diseases.     

Alternative stabilities of a proline-rich antibacterial peptide in vitro and in vivo

The proline-rich designer antibacterial peptide dimer A3-APO is currently under preclinical development for the treatment of systemic infections caused by antibiotic-resistant Gram-negative bacteria. The peptide showed remarkable stability in 25% mouse serum in vitro, exhibiting a half-life of approximately 100 min as documented by reversed-phase chromatography. Indeed, after a 30-min incubation period in undiluted mouse serum ex vivo, mass spectrometry failed to identify any degradation product. The peptide was still a major peak in full blood ex vivo, however, with degradation products present corresponding to amino-terminal cleavage. When injected into mice intravenously, very little, if any unmodified peptide could be detected after 30 min. Nevertheless, the major early metabolite, a full single-chain fragment, was detectable until 90 min, and this fragment exhibited equal or slightly better activity in the broth microdilution antimicrobial assay against a panel of resistant Enterobactericeae strains. The Chex1-Arg20 metabolite, when administered three times at 20 mg/kg to mice infected with a sublethal dose (over LD(50)) of an extended spectrum beta-lactamase-producing Escherichia coli strain, completely sterilized the mouse blood, similar to imipenem added at a higher dose. The longer and presumably more immunogenic prodrug A3-APO, injected subcutaneously twice over a 3-wk period, did not induce any antibody production, indicating the suitability of this peptide or its active metabolite for clinical development.     

Identification of crucial residues for the antibacterial activity of the proline-rich peptide, pyrrhocoricin.

Members of the proline-rich antibacterial peptide family, pyrrhocoricin, apidaecin and drosocin appear to kill responsive bacterial species by binding to the multihelical lid region of the bacterial DnaK protein. Pyrrhocoricin, the most potent among these peptides, is nontoxic to healthy mice, and can protect these animals from bacterial challenge. A structure-antibacterial activity study of pyrrhocoricin against Escherichia coli and Agrobacterium tumefaciens identified the N-terminal half, residues 2-10, the region responsible for inhibition of the ATPase activity, as the fragment that contains the active segment. While fluorescein-labeled versions of the native peptides entered E. coli cells, deletion of the C-terminal half of pyrrhocoricin significantly reduced the peptide's ability to enter bacterial or mammalian cells. These findings highlighted pyrrhocoricin's suitability for combating intracellular pathogens and raised the possibility that the proline-rich antibacterial peptides can deliver drug leads into mammalian cells. By observing strong relationships between the binding to a synthetic fragment of the target protein and antibacterial activities of pyrrhocoricin analogs modified at strategic positions, we further verified that DnaK was the bacterial target macromolecule. Inaddition, the antimicrobial activity spectrum of native pyrrhocoricin against 11 bacterial and fungal strains and the binding of labeled pyrrhocoricin to synthetic DnaK D-E helix fragments of the appropriate species could be correlated. Mutational analysis on a synthetic E. coli DnaK fragment identified a possible binding surface for pyrrhocoricin.     

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.     

Structure-function study of cathelicidin-derived bovine antimicrobial peptide BMAP-28: Design of its cell-selective analogs by amino acid substitutions in the heptad repeat sequences

Although BMAP-28 is a potent cathelicidin-derived bovine antimicrobial peptide, its cytotoxic activity against the human and other mammalian cells is of concern for converting it into a novel antimicrobial drug. We have identified a short leucine and isoleucine zipper sequences at the N- and C-terminals of BMAP-28, respectively. To understand the possible role of these structural elements in BMAP-28, a number of alanine-substituted analogs were designed, synthesized and characterized along with the wild-type peptide. The substitution of amino acids at single or multiple ‘a’ position(s) of these structural motifs by alanine showed significant effects on the cytotoxic activity of the molecule on the human red blood cells (hRBCs) and 3T3 cells without showing much effects on their MIC values against the selected bacteria. BMAP-28 and all its analogs depolarized the Escherichia coli cells with almost equal efficacy. In contrast, the alanine-substituted analogs of BMAP-28 depolarized hRBCs much less efficiently than the parent molecule. Results further showed that BMAP-28 assembled appreciably onto the live E. coli and hRBC. However, the selected less toxic analogs of BMAP-28 although assembled as good as the parent molecule onto the live E. coli cells, their assembly onto the live mammalian hRBCs was much weaker as compared to that of the wild-type molecule. Looking at the remarkable similarity with the data presented in our previous work on melittin, it appears that probably the heptad repeat sequence possesses a general role in maintaining the cytotoxicity of the antimicrobial peptides against the mammalian cells and assembly therein.     

Structure-activity relationships of diastereomeric lysine ring size analogs of the antimicrobial peptide gramicidin S: mechanism of action and discrimination between bacterial and animal cell membranes

Structure-activity relationships were examined in seven gramicidin S analogs in which the ring-expanded analog GS14 [cyclo-(VKLKVdYPLKVKLdYP)] is modified by enantiomeric inversions of its lysine residues. The conformation, amphiphilicity, and self-association propensity of these peptides were investigated by circular dichroism spectroscopy and reversed phase high performance liquid chromatography. (31)P nuclear magnetic resonance spectroscopic and dye leakage experiments were performed to evaluate the capacity of these peptides to induce inverse nonlamellar phases in, and to permeabilize phospholipid bilayers; their growth inhibitory activity against the cell wall-less mollicute Acholeplasma laidlawii B was also examined. The amount and stability of beta-sheet structure, effective hydrophobicity, propensity for self-association in water, ability to disrupt the organization of phospholipid bilayers, and ability to inhibit A. laidlawii B growth are strongly correlated with the facial amphiphilicity of these GS14 analogs. Also, the magnitude of the parameters segregate these peptides into three groups, consisting of GS14, the four single inversion analogs, and the two multiple inversion analogs. The capacity of these peptides to differentiate between bacterial and animal cell membranes exhibits a biphasic relationship with peptide amphiphilicity, suggesting that there may only be a narrow range of peptide amphiphilicity within which it is possible to achieve the dual therapeutic requirements of high antibiotic effectiveness and low hemolytic activity. These results were rationalized by considering how the physiochemical properties of these GS14 analogs are likely to be reflected in their partitioning into lipid bilayer membranes.     

Design and synthesis of triple inhibitors of janus kinase (JAK), histone deacetylase (HDAC) and Heat Shock Protein 90 (HSP90)

Inhibition of multiple signaling pathways in a cancer cell with a single molecule could result in better therapies that are simpler to administer. Efficacy may be achieved with reduced potency against individual targets if there is synergy through multiple pathway inhibition. To achieve this, it is necessary to be able to build multi-component ligands by joining together key pharmacophores in a way which maintains sufficient activity against the individual pathways. In this work, designed triple inhibiting ligands are explored aiming to block three completely different target types: a kinase (JAK2), an epigenetic target (HDAC) and a chaperone (HSP90). Although these enzymes have totally different functions they are related through inter-dependent pathways in the developing cancer cell. Synthesis of several complex multi-inhibiting ligands are presented along with initial enzyme inhibition data against 3 biological target classes of interest. A lead compound, 47, was discovered which had low micromolar activity for all 3 targets. Further development of these complex trispecific designed multiple ligands could result in a ‘transient drug’, an alternative combination therapy for treating cancer mediated via a single molecule.     

Synthesis and antimicrobial activity of cysteine-free coprisin nonapeptides

Coprisin is a 43-mer defensin-like peptide from the dung beetle, Copris tripartitus. CopA3 (LLCIALRKK-NH₂), a 9-mer peptide containing a single free cysteine residue at position 3 of its sequence, was derived from the α-helical region of coprisin and exhibits potent antibacterial and anti-inflammatory activities. The single cysteine implies a tendency for dimerization; however, it remains unknown whether this cysteine residue is indispensible for CopA3’s antimicrobial activity. To address this issue, in the present study we synthesized eight cysteine-substituted monomeric CopA3 analogs and two dimeric analogs, CopA3 (Dimer) and CopIK (Dimer), and evaluated their antimicrobial effects against bacteria and fungi, as well as their hemolytic activity toward human erythrocytes. Under physiological conditions, CopA3 (Mono) exhibits a 6/4 (monomer/dimer) molar ratio in HPLC area percent, indicating that its effects on bacterial strains likely reflect a CopA3 (Mono)/CopA3 (Dimer) mixture. We also report the identification of CopW, a new cysteine-free nonapeptide derived from CopA3 that has potent antimicrobial activity with virtually no hemolytic activity. Apparently, the cysteine residue in CopA3 is not essential for its antimicrobial function. Notably, CopW also exhibited significant synergistic activity with ampicillin and showed more potent antifungal activity than either wild-type coprisin or melittin.     

Enhancement of the anti‐inflammatory activity of temporin‐1Tl‐derived antimicrobial peptides by tryptophan,arginine and lysine substitutions

Temporin‐1Tl (TL) is a 13‐residue frog antimicrobial peptide (AMP) exhibiting potent antimicrobial and anti‐inflammatory activity. To develop novel AMP with improved anti‐inflammatory activity and antimicrobial selectivity, we designed and synthesized a series of TL analogs by substituting Trp, Arg and Lys at selected positions. Except for Escherichia coli and Staphylococcus epidermidis, all TL analogs exhibited retained or increased antimicrobial activity against seven bacterial strains including three methicillin‐resistant Staphylococcus aureus strains compared with TL. TL‐1 and TL‐4 showed a little increase in antimicrobial selectivity, while TL‐2 and TL‐3 displayed slightly decreased antimicrobial selectivity because of their about twofold increased hemolytic activity. All TL analogs demonstrated greatly increased anti‐inflammatory activity, evident by their higher inhibition of the production tumor necrosis factor‐α (TNF‐α) and nitric oxide and the mRNA expression of inducible nitric oxide synthase and TNF‐α in lipopolysaccharide (LPS)‐stimulated RAW264.7 macrophage cells, compared with TL. Taken together, the peptide anti‐inflammatory activity is as follows: TL‐2 ≈ TL‐3 ≈ TL‐4 > TL‐1 > TL. In addition, LPS binding ability of the peptides corresponded with their anti‐inflammatory activity. These results apparently suggest that the anti‐inflammatory activity of TL analogs is associated with the direct binding ability between these peptides and LPS. Collectively, our designed TL analogs possess improved anti‐inflammatory activity and retain antimicrobial activity without a significant increase in hemolysis. Therefore, it is evident that our TL analogs constitute promising candidates for the development of peptide therapeutics for gram‐negative bacterial infection. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Lasiocepsin, a novel cyclic antimicrobial peptide from the venom of eusocial bee Lasioglossum laticeps (Hymenoptera: Halictidae)

In the venom of eusocial bee Lasioglossum laticeps, we identified a novel unique antimicrobial peptide named lasiocepsin consisting of 27 amino acid residues and two disulfide bridges. After identifying its primary structure, we synthesized lasiocepsin by solid-phase peptide synthesis using two different approaches for oxidative folding. The oxidative folding of fully deprotected linear peptide resulted in a mixture of three products differing in the pattern of disulfide bridges. Regioselective disulfide bond formation significantly improved the yield of desired product. The synthetic lasiocepsin possessed antimicrobial activity against both Gram-positive and -negative bacteria, antifungal activity against Candida albicans, and no hemolytic activity against human erythrocytes. We synthesized two lasiocepsin analogs cyclized through one native disulfide bridge in different positions and having the remaining two cysteines substituted by alanines. The analog cyclized through a Cys8-Cys25 disulfide bridge showed reduced antimicrobial activity compared to the native peptide while the second one (Cys17-Cys27) was almost inactive. Linear lasiocepsin having all four cysteine residues substituted by alanines or alkylated was also inactive. That was in contrast to the linear lasiocepsin with all four cysteine residues non-paired, which exhibited remarkable antimicrobial activity. The shortening of lasiocepsin by several amino acid residues either from the N- or C-terminal resulted in significant loss of antimicrobial activity. Study of Bacillus subtilis cells treated by lasiocepsin using transmission electron microscopy showed leakage of bacterial content mainly from the holes localized at the ends of the bacterial cells.     

Chemical synthesis, molecular modeling, and antimicrobial activity of a novel bacteriocin, MMFII

A new antimicrobial peptide, referred to as MMFII, was purified to homogeneity from lactic acid bacteria Lactococcus lactis, which were isolated from Tunisian dairy product. The complete amino acid sequence of the peptide has been established by amino acid analysis, Edman sequencing, and mass spectrometry and verified by solid-phase chemical synthesis. MMFII is a single-chain 37-residue polypeptide containing a single intramolecular disulfide bond, i.e., TSYGNGVHCNKSKCWIDVSELETYKAGTVSNPKDILW. It shares ca. 35% sequence identity with Leucocin A, a class IIa bacteriocin. Modeling based on the 3-D of Leucocin A shows three beta strands located in the N-terminal region (Thr1-Tyr3, Val7-Asn10, Lys13-Ile16) and an alpha helical domain from Asp17 to Asn31. When plotted as an alpha-helical wheel, the central alpha-helix of MMFII does not exhibit an amphipathic helical structure. The synthetic MMFII (sMMFII), obtained by the solid-phase method, was shown to be indistinguishable from the natural peptide. sMMFII is active against Lactococcus cremoris and Listeria ivanovii bacteria, whereas no activity was detected for any of the synthetic N-terminal truncated MMFII analogs Cys9-Trp37, Trp15-Trp37, and Val18-Trp37.     

Structure-activity relationship of HP (2-20) analog peptide: enhanced antimicrobial activity by N-terminal random coil region deletion

HP (2-20) (AKKVFKRLEKLFSKIQNDK) is a 19-aa antimicrobial peptide derived from N-terminus of Helicobacter pylori Ribosomal protein L1 (RpL1). In the previous study, several analogs with amino acid substitutions were designed to increase or decrease only the net hydrophobicity. In particular, substitutions of Gln(16) and Asp(18) with Trp (Anal 3) for hydrophobic amino acid caused a dramatic increase in antibiotic activity without a hemolytic effect. HP-A3 is a potent antimicrobial peptide that forms, in a hydrophobic medium, an amphipathic structure consisting of an N-terminal random coil region (residues 2-5) and extended C-terminal regular alpha-helical region (residues 6-20). To obtain the short and potent alpha-helical antimicrobial peptide, we synthesized a N-terminal random coil deleted HP-A3 (A3-NT) and examined their antimicrobial activity and mechanism of action. The resulting 15mer peptide showed increased antibacterial and antifungal activity to 2- and 4-fold, respectively, without hemolysis. Confocal fluorescence microscopy studies showed that A3-NT was accumulated in the plasma membrane. Flow cytometric analysis revealed that A3-NT acted in salt- and energy-independent manner. Furthermore, A3-NT causes significant morphological alterations of the bacterial surfaces as shown by scanning electron microscopy. Circular dichroism (CD) analysis revealed that A3-NT showed higher alpha-helical contents than the HP-A3 peptide in 50% TFE solution. Therefore, the cell-lytic efficiency of HP-A3, which depended on the alpha-helical content of peptide, correlated linearly with their antimicrobial potency.     

Formation of ion channels in planar lipid bilayer membranes by synthetic basic peptides.

We made use of a planar lipid bilayer system to examine the action of synthetic basic peptides which model the prepiece moiety of mitochondrial protein precursors and have antibacterial activity against Gram-positive bacteria. The sequences of the peptides used were as follows: Ac-(Ala-Arg-Leu)3-NHCH3 (3(3], Ac-(Leu-Ala-Arg-Leu)2-NHCH3 (4(2], Ac-(Leu-Ala-Arg-Leu)3-NHCH3 (4(3], Ac-(Leu-Leu-Ala-Arg-Leu)2-NHCH3 (5(2]. These peptides interacted differently with planar lipid bilayer membranes and membrane conductance increased by the formation of ion channels. The effects of the peptides on the macroscopic current-increase and on the probability of channel formation, at the single channel level were in the order of 4(3) greater than 4(2) approximately 5(2) much greater than 3(3), a finding which correlates with the antibacterial activity of these peptides. The micromolar (microM) order concentration at which the channel was formed resembles that causing antibacterial activity. Thus, the peptide antibacterial activity may occur through an increase in ion permeability of the bacterial membrane. The single-channel properties were investigated in detail using 4(3), the peptide with the highest ion channel-forming activity. Many types of channels were observed with respect to conductance (2-750 pS) and voltage dependency of gating. However, the channels were all cation-selective. These results suggest that the ion channels formed by peptide 4(3) may be able to take on a variety of conformations and/or assembly.     

The effects of ring-size analogs of the antimicrobial peptide gramicidin S on phospholipid bilayer model membranes and on the growth of Acholeplasma laidlawii B.

We have examined the effects of three ring-size analogs of the cyclic beta-sheet antimicrobial peptide gramicidin S (GS) on the thermotropic phase behavior and permeability of phospholipid model membranes and on the growth of the cell wall-less Gram-positive bacteria Acholeplasma laidlawii B. These three analogs have ring sizes of 10 (GS10), 12 (GS12) or 14 (GS14) amino acids, respectively. Our high-sensitivity differential scanning calorimetric studies indicate that all three of these GS analogs perturb the gel/liquid-crystalline phase transition of zwitterionic phosphatidylcholine (PtdCho) vesicles to a greater extent than of zwitterionic phosphatidylethanolamine (PtdEtn) or of anionic phosphatidylglycerol (PtdGro) vesicles, in contrast to GS itself, which interacts more strongly with PtdGro than with PtdCho and PtdEtn bilayers. However, the relative potency of the perturbation of phospholipid phase behavior varies markedly between the three peptides, generally decreasing in the order GS14 > GS10 > GS12. Similarly, these three GS ring-size analogs also differ considerably in their ability to cause fluorescence dye leakage from phospholipid vesicles, with the potency of permeabilization also generally decreasing in the order GS14 > GS10 > GS12. Finally, these GS ring-size analogs also differentially inhibit the growth of A. laidlawii with growth inhibition also decreasing in the order GS14 > GS10 > GS12. These results indicate that the relative potencies of GS and its ring-size analogs in perturbing the organization and increasing the permeability of phospholipid bilayer model membranes, and of inhibiting the growth of A. laidlawii B cells, are at least qualitatively correlated, and provide further support for the hypothesis that the primary target of these antimicrobial peptides is the lipid bilayer of the bacterial membrane. The very high antimicrobial activity of GS14 against the cell wall-less bacteria A. laidlawii as compared to various conventional bacteria confirms our earlier suggestion that the avid binding of this peptide to the bacterial cell wall is primarily responsible for its reduced antimicrobial activity against such organisms. The relative magnitude of the effects of GS itself, and of the three ring-size GS analogs, on phospholipid bilayer organization and cell growth correlate relatively well with the effective hydrophobicities and amphiphilicities of these peptides but less well with their relative charge density, intrinsic hydrophobicities or conformational flexibilities. Nevertheless, all of these parameters, as well as others, may influence the antimicrobial potency and hemolytic activity of GS analogs.     

RNA interference of an antimicrobial peptide, gloverin, of the beet armyworm, Spodoptera exigua, enhances susceptibility to Bacillus thuringiensis

Gloverin is known to be an inducible antimicrobial peptide. This study reports a gloverin gene (Seglv) identified from the beet armyworm, Spodoptera exigua. Seglv encodes 175 amino acids with a signal peptide. Its amino acid sequence is highly homologous (>95%) to other known gloverins. Seglv was expressed from egg to adult stages even without immune challenge. Especially, in larval stage, it was expressed in all tested tissues, such as hemocyte, fat body, gut, and epidermis. However, the constitutive expression level was significantly elevated in response to bacterial challenge. Expression of a Toll gene was required for expression of Seglv. A recombinant Seglv protein was synthesized using a bacterial expression system and purified with an affinity chromatography. The recombinant protein showed a specific antibacterial activity against a Gram-positive bacterium, but no activity against a Gram-negative Escherichia coli. Injection of specific double stranded RNA (dsRNA) against Seglv could suppress its expression. Knockdown of Seglv expression induced a significant developmental retardation and resulted in hypotrophy pupae. The larvae treated with dsRNA were much more susceptible to Bacillus thuringiensis than the control larvae. These results suggest that Seglv acts as an antimicrobial peptide especially against Gram-positive bacteria including B. thuringiensis.     

中国明对虾溶菌酶基因克隆、重组表达与性质分析

溶菌酶是机体先天免疫系统中一个重要的效应分子, 参与机体多种免疫反应, 在溶菌过程中形成一个水解体系, 破坏和消除侵入体内的病原, 从而实现机体的免疫防御。从中国明对虾中克隆得到了溶菌酶基因(称为FcLyz基因), 该基因全长709 bp, 其完整的阅读框为477 bp, 编码158个氨基酸, 前18个氨基酸(-1~-18)为信号肽, 成熟肽由140个氨基酸组成(1-140aa), 其分子量为16.2 kD。经SMART分析,该基因具有1个溶菌酶1(LYZ1)结构域(19-130aa)。半定量RT-PCR分析结果表明溶菌酶虽在多种组织中有较低水平的组成性表达, 但在细菌诱导的血细胞、心脏、肝胰腺和鳃等多种组织中表达上调。将中国明对虾溶菌酶基因的成熟肽亚克隆进原核表达载体pET-30a (+)中, 转化大肠杆菌BL21(DE3), 再进行诱导表达和亲和纯化, 得到了纯化的重组溶菌酶, 并进行了抑菌活性检测。结果表明, 重组对虾溶菌酶对革兰氏阳性菌的抑菌能力较强, 最小抑菌浓度达到3.43 mmol/L, 但对革兰氏阴性菌抑制作用较小。上述结果表明, 该溶菌酶作为一种重要的免疫效应分子, 参与了对虾的免疫防御反应。     

中国明对虾溶菌酶基因克隆、重组表达与性质分析

溶菌酶是机体先天免疫系统中一个重要的效应分子, 参与机体多种免疫反应, 在溶菌过程中形成一个水解体系, 破坏和消除侵入体内的病原, 从而实现机体的免疫防御。从中国明对虾中克隆得到了溶菌酶基因(称为FcLyz基因), 该基因全长709 bp, 其完整的阅读框为477 bp, 编码158个氨基酸, 前18个氨基酸(-1~-18)为信号肽, 成熟肽由140个氨基酸组成(1-140aa), 其分子量为16.2 kD。经SMART分析,该基因具有1个溶菌酶1(LYZ1)结构域(19-130aa)。半定量RT-PCR分析结果表明溶菌酶虽在多种组织中有较低水平的组成性表达, 但在细菌诱导的血细胞、心脏、肝胰腺和鳃等多种组织中表达上调。将中国明对虾溶菌酶基因的成熟肽亚克隆进原核表达载体pET-30a (+)中, 转化大肠杆菌BL21(DE3), 再进行诱导表达和亲和纯化, 得到了纯化的重组溶菌酶, 并进行了抑菌活性检测。结果表明, 重组对虾溶菌酶对革兰氏阳性菌的抑菌能力较强, 最小抑菌浓度达到3.43 mmol/L, 但对革兰氏阴性菌抑制作用较小。上述结果表明, 该溶菌酶作为一种重要的免疫效应分子, 参与了对虾的免疫防御反应。