Expression of the recombinant antibacterial peptide sarcotoxin IA in Escherichia coli cells

Sarcotoxin IA is an antibacterial peptide that is secreted by a meat-fly Sarcophaga peregrina larva in response to a hypodermic injury or bacterial infection. This peptide is highly toxic against a broad spectrum of both Gram-positive and Gram-negative bacteria and lethal to microbes even at nanomolar concentrations. However, research needs as well as its potential use in medicine require substantial amounts of highly purified sarcotoxin. Because heterologous expression systems proved to be inefficient due to sarcotoxin sensitivity to intracellular proteases, here we propose the biosynthesis of sarcotoxin precursors in Escherichia coli cells that are highly sensitive to the mature peptide. To optimize its biosynthesis, sarcotoxin was translationally fused with proteins highly expressed in E. coli. A fusion partner and the position of sarcotoxin in the chimeric polypeptide were crucial for protecting the sarcotoxin portion of the fusion protein from proteolysis. Released after chemical cleavage of the fusion protein and purified to homogeneity, sarcotoxin displayed antibacterial activity comparable to that previously reported for the natural peptide.     

Induced expression of sarcotoxin IA enhanced host resistance against both bacterial and fungal pathogens in transgenic tobacco

We demonstrate here that induced expression of sarcotoxin IA, a bactericidal peptide from Sarcophaga peregrina, enhanced the resistance of transgenic tobacco plants to both bacterial and fungal pathogens. The peptide was produced with a modified PR1a promoter, which is further activated by salicylic acid treatment and necrotic lesion formation by pathogen infection. Host resistance to infection of bacteria Erwinia carotovora subsp. carotovora and Pseudomonas syringae pv. tabaci was shown to be dependent on the amounts of sarcotoxin IA expressed. Since we found antifungal activity of the peptide in vitro, transgenic seedlings were also inoculated with fungal pathogens Rhizoctonia solani and Pythium aphanidermatum. Transgenic plants expressing higher levels of sarcotoxin were able to withstand fungal infection and remained healthy even after 4 weeks, while control plants were dead by fungal infection after 2 weeks.     

Synthesis and antibacterial activity of analogues of the N-terminal fragment of the sarcotoxin IA antimicrobial peptide

Three 18-membered analogues of the N-terminal fragment of the sarcotoxin IA cationic antimicrobial peptide were synthesized by the solid phase method of peptide synthesis with the use of swellographic monitoring. The ability of these peptides to inhibit the growth of various bacteria in culture medium and their hemolytic activity in experiments on human erythrocytes were studied. The analogue completely corresponding to the N-terminal amino acid sequence of the natural sarcotoxin IA with the amide group on its C-terminus exhibited higher antibacterial activity. The presence of carboxyl group on the C-terminus or the substitution of Tyr for Trp2 resulted in a decrease in the antimicrobial activity of the peptide. Our results indicate that the amphiphilic N-terminal peptide corresponding to the 1-18 sequence of sarcotoxin IA involves the moieties responsible for the antimicrobial activity of the antibiotic.     

Production of recombinant sarcotoxin IA in Bombyx mori cells.

A cDNA for sarcotoxin IA, an antibacterial protein of Sarcophaga peregrina (fleshfly), was inserted into a silkworm baculovirus vector and expressed in Bm-N cells, a line of Bombyx mori cells. When a cysteine proteinase inhibitor, p-chloromercuribenzenesulphonic acid, was present in the culture medium, a significant amount of recombinant sarcotoxin IA accumulated, but without this reagent the product seemed to be degraded in this system. The C-terminus of the recombinant sarcotoxin IA seemed to be glycine, not amidated arginine as found in authentic sarcotoxin IA. Probably, Bm-N cells lack the C-terminal alpha-amidation enzyme.     

Synthesis and Antibacterial Activity of Analogues of the N-Terminal Fragment of the Sarcotoxin IA Antimicrobial Peptide

Three 18-membered analogues of the N-terminal fragment of the sarcotoxin IA cationic antimicrobial peptide were synthesized by the solid phase method of peptide synthesis with the use of swellographic monitoring. The ability of these peptides to inhibit the growth of various bacteria in culture medium and their hemolytic activity in experiments on human erythrocytes were studied. The analogue completely corresponding to the N-terminal amino acid sequence of the natural sarcotoxin IA with the amide group on its C-terminus exhibited higher antibacterial activity. The presence of carboxyl group on the C-terminus or the substitution of Tyr for Trp2 resulted in a decrease in the antimicrobial activity of the peptide. Our results indicate that the amphiphilic N-terminal peptide corresponding to the 1–18 sequence of sarcotoxin IA involves the moieties responsible for the antimicrobial activity of the antibiotic.     

Interaction between liposomes and sarcotoxin IA, a potent antibacterial protein of Sarcophaga peregrina (flesh fly)

The direct interaction between phospholipids and sarcotoxin IA, a potent bactericidal protein of Sarcophaga peregrina, was studied using authentic sarcotoxin IA, its synthetic derivatives, and various liposomes. Results showed that sarcotoxin IA interacted with liposomes constituted from acidic phospholipids, resulting in the release of glucose trapped in these liposomes. The amidated carboxyl-terminal of this protein was found to be important for this interaction. Liposomes constituted from total phospholipids of Escherichia coli became less susceptible to sarcotoxin IA with an increase in their cholesterol content. Since bacterial membranes do not contain cholesterol, this finding may partly explain the selective toxicity of sarcotoxin I to bacteria.     

Participation of two N-terminal residues in LPS-neutralizing activity of sarcotoxin IA

Sarcotoxin IA is a cecropin-type antibacterial peptide of flesh fly. Using a mutant sarcotoxin IA lacking two N-terminal residues, we demonstrated that these residues are indispensable for its antibacterial activity against Escherichia coli and LPS-binding. Contrary to the native sarcotoxin IA, the mutant sarcotoxin IA could not neutralize various biological activities of LPS. It was suggested that sarcotoxin IA firmly binds to the lipid A core of LPS via these two N-terminal residues and forms a stable binding complex that exhibits no appreciable biological activity like native LPS.     

Primary structure of sarcotoxin I, an antibacterial protein induced in the hemolymph of Sarcophaga peregrina (flesh fly) larvae

The primary structure of sarcotoxin I, a potent bactericidal protein induced in the hemolymph of larvae of Sarcophaga peregrina (flesh fly), was investigated. Sarcotoxin I was a mixture of three proteins (sarcotoxins IA, IB, and IC) with almost identical primary structures. These proteins were found to consist of 39 amino acid residues and to differ in only 2-3 amino acid residues. The amino-terminal half of the molecules was rich in charged amino acids and was hydrophilic, whereas the carboxyl-terminal half was hydrophobic. It is suggested that the carboxyl-terminal half of sarcotoxin I penetrates into the bacterial membrane and that its amino-terminal half rich in basic amino acid residues interacts with acidic phospholipids in the bacterial membrane, resulting in perturbation of the membrane and loss of viability of the bacteria.     

Molecular cloning of a cDNA and assignment of the C-terminal of sarcotoxin IA, a potent antibacterial protein of Sarcophaga peregrina.

A previous paper described the complete amino acid sequences of sarcotoxins IA, IB and IC, which are a group of potent antibacterial proteins with almost identical primary structures produced by Sarcophaga peregrina (fleshfly) larvae [Okada & Natori (1985) J. Biol. Chem. 260, 7174-7177]. The present paper describes the cDNA cloning and complete nucleotide sequencing of a cDNA clone for sarcotoxin IA. The C-terminal amino acid residue of sarcotoxin IA deduced from the nucleotide sequence was glycine, whereas it was found to be arginine by amino acid sequencing of purified sarcotoxin IA. Analysis of the elution profiles on h.p.l.c. of the synthetic derivatives of sarcotoxin IA showed that the C-terminal amino acid residue of authentic sarcotoxin IA is amidated arginine, which is probably produced by enzymic cleavage of terminal glycine.     

Purification of three antibacterial proteins from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina

Three antibacterial proteins were purified from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina (flesh fly). Sequencing studies showed that two of these proteins belong to the sarcotoxin I family, potent antibacterial proteins purified from the hemolymph of Sarcophaga larvae, whereas the other protein, named sapecin, is a new protein consisting of 40 amino acid residues including 6 cysteine residues. Unlike sarcotoxin I, sapecin preferentially represses the growth of various Gram-positive bacteria. The proteins of the sarcotoxin I family produced by this cell line were found to have carboxyl-terminal glycine, whereas sarcotoxin I in the hemolymph has amidated amino acids. This suggests that the embryonic cells lack an enzyme that cleaves off carboxyl-terminal glycine to form a new amidated carboxyl terminus.     

Expression of a gene encoding a scorpion insectotoxin peptide in yeast, bacteria and plants.

The nucleotide sequence encoding the scorpion insectotoxin I5A was chemically synthesized and expressed in yeast, bacteria and tobacco. The I5A peptides produced in these organisms were purified using an immunoaffinity chromatography procedure. I5A produced using the bacterial secretion system was efficiently secreted and released into the culture medium. In contrast, only a trace amount of I5A was detected in bacterial cytosols when expressed from a direct expression vector, suggesting that I5A was unstable in bacterial cells. I5A secreted from yeast using an alpha-factor signal sequence was shown to have an N-terminal (Glu-Ala)2 extension, indicating incomplete processing of the secreted peptide by dipeptidyl aminopeptidase A. In tobacco, a nonsecreted form of the protein was produced. No measurable insect toxicity was observed when insect larvae were assayed, regardless of whether I5A was produced in yeast, bacteria or tobacco. The lack of toxicity is almost certainly the result of improper folding due to incorrect disulfide bond formation. The inability to produce a biologically active peptide must be overcome before scorpion toxins might be used for the genetic engineering of plants for insect resistance. The yeast and bacterial expression systems described here may be useful for further studies on the problem of expressing a biologically active peptide.     

A Novel Antimicrobial Peptide Derived from the Insect Paederus dermatitis

Much research has been focused on antimicrobial peptides (AMPs) derived from insect immune defense reactions due to their potential in the development of new antibiotics. In this study, a new AMP from the insect Paederus dermatitis, named sarcotoxin Pd was identified and purified using gel filtration and reverse-phase high-performance liquid chromatography. Our results showed that this peptide has broad-spectrum inhibitory effects on examined microbes. Sarcotoxin Pd is composed of 34 amino acids and its molecular weight was estimated to be 3613.26 ± 0.5 Da. Minimum inhibitory concentration (MIC) values of sarcotoxin Pd against Gram-negative bacteria were lower than Gram-positive bacteria and fungi. The identified peptide showed the highest antimicrobial effect against Klebsiella pneumonia and Escherichia coli. This peptide did not reveal significant hemolytic activity against human red blood cells particularly in the range of MIC values. Confirming the potential antimicrobial activities of synthetic peptide, this paper addresses the role of sarcotoxin Pd in the treatment of systemic microbial illnesses.     

A peptide from insects protects transgenic tobacco from a parasitic weed

Parasitic plants present some of the most intractable weed problems for agriculture in much of the world. Species of root parasites such as Orobanche can cause enormous yield losses, yet few control measures are effective and affordable. An ideal solution to this problem is the development of parasite-resistant crops, but this goal has been elusive for most susceptible crops. Here we report a mechanism for resistance to the parasitic angiosperm Orobanche based on expression of sarcotoxin IA in transgenic tobacco. Sarcotoxin IA is a 40-residue peptide with antibiotic activity, originally isolated from the fly, Sarcophaga peregrina. The sarcotoxin IA gene was fused to an Orobanche-inducible promoter, HMG2, which is induced locally in the host root at the point of contact with the parasite, and used to transform tobacco. The resulting transgenic plants accumulated more biomass than non-transformed plants in the presence of parasites. Furthermore, plants expressing sarcotoxin IA showed enhanced resistance to O. aegyptiaca as evidenced by abnormal parasite development and higher parasite mortality after attachment as compared to non-transformed plants. The transgenic plants were similar in appearance to non-transformed plants suggesting that sarcotoxin IA is not detrimental to the host.     

Structure-activity relationships of de novo designed cyclic antimicrobial peptides based on gramicidin S

The cyclic beta-sheet structure possessed by the 10-residue antibiotic peptide gramicidin S was taken as the structural framework for the de novo design of biologically active peptides with membrane-active properties. We have shown from previous studies that gramicidin S is a broad-spectrum antibiotic effective against Gram-positive bacteria, Gram-negative bacteria, and fungi, but is toxic to human red blood cells. We tested the effect of ring size on antimicrobial activity and hemolytic activity on peptides varying from 4 to 16 residues. Interestingly, we were able to dissociate hemolytic activity and antimicrobial activity by increasing the ring size of the peptide to 14 residues (peptide GS14). Furthermore, we increased specificity for microbial membranes while decreasing toxicity to red blood cells by substituting enantiomers (D-amino acids for L-amino acids and vice versa) into the GS14 sequence. The enantiomeric substitutions all disrupted beta-sheet structure in benign medium and decreased peptide amphipathicity. The least amphipathic peptide, produced by substituting a D-Lys at position 4 of GS14 (peptide GS14K4), also had the highest therapeutic index, i.e., highest degree of specificity for microbial cells over human cells. Solution structures of GS14 analogs solved by NMR spectroscopy showed that the D-amino acid side chain was located on the nonpolar face of GS14K4. Another analog, a beta-sheet peptide with reduced amphipathicity (peptide GS14 K3L4), also had a lysine (lysine 3) on the nonpolar face as determined by the NMR structure. Both GS14K4 and GS14 K3L4 had reduced amphipathicity relative to GS14 and much higher therapeutic indices. Finally, the alteration of the nonpolar face hydrophobicity of GS14K4 analogs provided a range of activities and specificities, where the peptides with the intermediate hydrophobicities among the series had the highest therapeutic indices. The optimal peptide hydrophobicities varied depending on the microorganism being tested, with higher hydrophobicity requirements against Gram-positive bacteria and yeast compared with Gram-negative microorganisms. The net result of these studies suggests that it is possible to rationally design a cyclic membrane-active antimicrobial peptide with high specificity towards prokaryotic (bacterial and fungal) membranes and minimal toxicity to eukaryotic (e.g., mammalian) membranes.     

Inhibitory effect of sarcotoxin IIA, an antibacterial protein of Sarcophaga peregrina, on growth of Escherichia coli

The effect of sarcotoxin IIA, an antibacterial protein of Sarcophaga peregrina (flesh fly), on Escherichia coli was investigated. Sarcotoxin IIA was found to have a bacterial effect on growing bacteria, but little on non-growing bacteria. At a concentration of 25 micrograms/ml, it induced significant morphological change of growing E. coli cells. In its presence, growing cells became greatly elongated, and spheroplast-like bulges and projections appeared on their surface. A rough mutant strain of E. coli with a defect in the structure of lipopolysaccharide was more sensitive than the parent strain to sarcotoxin IIA. These results suggest that the main effect of sarcotoxin IIA is to inhibit cell wall synthesis, including septum formation.     

Monoclonal antibodies against an HLA-B27-derived peptide react with an epitope present on bacterial proteins.

The role of molecular mimicry in the spondyloarthropathies was investigated with respect to the epitopes involved. mAb were produced against a synthetic peptide whose sequence was derived from a polymorphic region of the HLA-B27 molecule (amino acids 63-83). Two antibodies (J7F2 and H2B6) were selected for study on the basis of their ability to react with bacterial envelope proteins (ELISA) and B27-positive cells (immunofluorescence). J7F2 reacted preferentially with B27-positive cells and neither antibody reacted with MHC class I negative cells. Based on SDS-PAGE blot analysis of bacterial envelope proteins, the pattern of reactivity for both antibodies (against 36- and 19-kDa proteins) was the same as that for a third monoclonal produced against bacterial envelope and reactive with B27-positive cells. This apparent epitope similarity was investigated by using synthetic peptides to inhibit binding of the monoclonals. The B27 synthetic peptide and a smaller peptide derived from it were efficient inhibitors of antipeptide and antibacterial antibody binding to bacterial Ag and B27-positive cells. These studies provide insight into the molecular basis of cross-reactivity between bacterial proteins and MHC class I molecules.     

Conventional laboratory agar media provide an iron-limited environment for bacterial growth

Abstract The outer membrane proteins of Escherichia coli and Pseudomonas aeruginosa grown in a number of conventional laboratory media were examined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) High-molecular-weight proteins similar to those produced by these strains in an iron-limited chemically defined medium were detected in cells grown on the surface of various agar media. In contrast, these proteins were not produced or were only poorly expressed by the corresponding broth cultures or by cells grown an agar supplemented with iron. A catecholic substance could be detected in DST agar extracts subsequent to bacterial growth which was produced to a lesser extent in IST agar and in broth cultures.     

粉纹夜蛾离体细胞抗菌肽的抗菌谱测定

用热灭活的大肠杆菌DHSQ诱导粉纹夜蛾(Trichoplusia ni)离体细胞产生抗菌肽,用三氯乙酸沉淀法提取出该活性物质,采用琼脂糖孔穴扩散法和生长抑制测定法测定其抗菌谱,发现该抗菌物质具有较广的抗微生物活性,其中特别是对革兰氏阴性菌中的沙门氏茵和大肠杆茵,酵母菌中的白色念珠菌,植物病源真茵中的花生白绢病茵和小麦赤霉病茵具有较强的抑菌活性,从而表明该物质是一种既抗细菌,又抗真菌的抗微生物肽。     

Induced immunity in Antheraea assama Ww larvae against flacherie causing Pseudomonas aeruginosa AC-3

This study reports for the first time the induction of immunity in Antheraea assama Ww larvae against bacterial flacherie. In silkworms group of disease caused by bacteria are collectively called "flacherie." This refers to the flaccid condition of the larvae due to the infections of bacterial strains pathogenic to muga silkworm. Antibacterial activity against pathogenic Pseudomonas aeruginosa AC-3 causing flacherie, was induced by injection of heat-killed cells of the same strain. Experiments on larval survivability and viable cell count revealed peak immune response on third day. Comparison of the amount of food ingested, excreta produced and larval weight of the saline-injected control, live bacteria-challenged larvae and heat-killed bacteria-injected larvae "(vaccinated)" confirmed the development of immunity against bacterial infection in the "vaccinated" set. The haemolymph of A. assama larvae was analyzed for proteins associated with bacterial infection. Out of the total 32 detected proteins, eleven (A1-2, A15-20, A22-23, and A29) were constitutively synthesized in both the control and live bacteria-injected larvae. Four inducible proteins A4, A9-10, and A21 were detected in the haemolymph of the live bacteria-injected larvae. Synthesis of rest of the proteins varied between the control and their live bacteria-injected counterparts. General protein profile of "vaccinated" larvae injected with live bacteria were found to be similar to that of the saline-injected control.