Expression of antimicrobial peptide Cecropin P1 in Saccharomyces cerevisiae and its antibacterial and antiviral activity in vitro

BACKGROUND: Cecropin P1, acting as an antimicrobial, has a broad-spectrum antibacterial activity with some antiviral and antifungal properties. It is a promising natural alternative to antibiotics which is originally isolated from the pig intestinal parasitic nematode Ascaris suum. Many studies have shown that Cecropin P1 is helpful for the prevention or treatment of clinical diseases. Therefore, it is very necessary to establish a safe, nontoxic, and efficient expression method of Cecropin P1. RESULTS: The results indicated that the recombinant protein was about 5.5 kDa showed by Tricine­SDS­ PAGE and Western blot. And Cecropin P1 was efficiently secreted and expressed after 12 h of induction, with an increasing yield over the course of the induction. Its maximum concentration was 7.83 mg/L after concentration and purification. In addition, in vitro experiments demonstrated that Cecropin P1 not only exerted a strong inhibitory effect on Escherichia coli, Salmonella sp., Shigella sp., and Pasteurella sp., but also displayed an antiviral activity against PRRSV NADC30-Like strain. CONCLUSIONS: Collectively, the strategy of expressing Cecropin P1 in Saccharomyces cerevisiae is harmless, efficient, and safe for cells. In addition, the expressed Cecropin P1 has antiviral and antibacterial properties concurrently.

Identification and recombinant expression of an antimicrobial peptide (cecropin B-like) from soybean pest Anticarsia gemmatalis

Insects can be found in numerous diverse environments, being exposed to pathogenic organisms like fungi and bacteria. Once these pathogens cross insect physical barriers, the innate immune system operates through cellular and humoral responses. Antimicrobial peptides are small molecules produced by immune signaling cascades that develop an important and generalist role in insect defenses against a variety of microorganisms. In the present work, a cecropin B-like peptide (AgCecropB) sequence was identified in the velvetbean caterpillar Anticarsia gemmatalis and cloned in a bacterial plasmid vector for further heterologous expression and antimicrobial tests. Methods AgCecropB sequence (without the signal peptide) was cloned in the plasmid vector pET-M30-MBP and expressed in the Escherichia coli BL21(DE3) expression host. Expression was induced with IPTG and a recombinant peptide was purified using two affinity chromatography steps with Histrap column. The purified peptide was submitted to high-resolution mass spectrometry (HRMS) and structural analyses. Antimicrobial tests were performed using gram-positive (Bacillus thuringiensis) and gram-negative (Burkholderia kururiensis and E. coli) bacteria. Results AgCecropB was expressed in E. coli BL21 (DE3) at 28°C with IPTG 0.5 mM. The recombinant peptide was purified and enriched after purification steps. HRMS confirmed AgCrecropB molecular mass (4.6 kDa) and circular dichroism assay showed α-helix structure in the presence of SDS. AgCrecropB inhibited almost 50% of gram-positive B. thuringiensis bacteria growth. Conclusions The first cecropin B-like peptide was described in A. gemmatalis and a recombinant peptide was expressed using a bacterial platform. Data confirmed tertiary structure as predicted for the cecropin peptide family. AgCecropB was capable to inhibit B. thuringiensis growth in vitro.(AU)

Antineoplastic mechanism of antimicrobial peptides: Selective membrane destruction and non-membrane dissolution / 中国组织工程研究

BACKGROUND: Antimicrobial peptides, an extract from nature, are a basic component of host immunity that make toxic effect on highly proliferative cells. They have attracted extensive attention of scientists. The understanding of the antineoplastic mechanism of antimicrobial peptides can contribute to its application in clinical practice. OBJECTIVE: To summarize the research advances in antineoplastic mechanism of antimicrobial peptides. METHODS: The first author conducted a computer-based retrieval of PubMed, Springerlink, Web of Science, and ScienceDirect databases for relevant articles published from January 2015 to May 2019. The keywords were “antimicrobial, peptide, antitumor mechanisms, antitumor activity and anti-neoplastic”. The articles concerning antineoplastic mechanism of antimicrobial peptides and research progress were selected. RESULTS AND CONCLUSION: Cationic antimicrobial peptides synthesized by ribosomes and the host defense peptides can interact with the membrane of bacteria, which showed a broad-spectrum antimicrobial activity. Compared with normal cells, the proportion of phosphatidylserine on the surface of cancer cells, which is negatively charged, is increased dramatically. As a result, the cationic amphiphilic peptides are good candidate for the antineoplastic drugs, and possess a high selectivity. There are two major antitumor mechanism of antimicrobial peptides, which are selective membrane destruction and non-membrane dissolution (α-defensin-1 and lactoferrin B). The clinical application of antimicrobial peptides against tumors is mainly restricted by their stability and the ways to administration. By optimizing its structure and drug delivery systems, these antimicrobial peptides will play a critical role in the cancer treatment.

Short chain linear and cyclic cationic peptide designed from cecropin B: Synthesis and anticancer activity

In the present work, we have designed short chain α-helical linear and cyclic peptide from cecropin B having samecharge, hydrophobicity, and helicity. The designed compounds were synthesized by using solution phase method.Elucidation of structure of newly synthesized peptides was done by proton nuclear magnetic resonance, Carbon-13nuclear magnetic resonance, Fourier-transform infrared spectroscopy, Fast atom bombardment mass spectrometry,and elemental analysis. Furthermore, the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltratrazolium bromide assay wasperformed for cytotoxicity of synthesized compounds against Dalton’s lymphoma ascites (DLA), Ehrlich’s ascitescarcinoma (EAC), and Michigan Cancer Foundation-7 cell lines using 5-FU as a reference compound. Biologicalevaluation showed that short chain cyclicpeptides were more potent than linear peptides against EAC and DLA celllines.

Study on expression and activity of combinant antimicrobial peptide in Pichia pastoris / 国际检验医学杂志

Objective To construct cecropin A-thanatin combinant gene engineering antimicrobial peptide gene CA(1-7)-T(4-19) for expression in Pichia pastoris.Methods The combinant antimicrobial peptide gene was artificially synthesized via gene splicing by overlap extension (SOE).The gene was cloned into the pPICZαA vector and transformed into Pichia pastoris X-33 by electroporation.The positive clones obtained by the screening of bleomycin resistance were induced by methanol ,and the antibacterial activity of the products was detected and the antimicrobial spectrum was established.Results The combinant peptide gene CA (1-7)-T (4-19) was successfully cloned on the carrier pPICZαA.The identification results were consistent with the pre-designed gene sequence.The combinant peptide gene was expressed under the induction of methanol ,and the minimum inhibitory concentration of 76 strains of Gram-egative and Gram-positive pathogenic bacteria isolated from the clinic was obtained ,and the minimum inhibitory concentration was up to 5 μg/mL.Conclusion A combinant genetic engineering antimicrobial peptide with antibacterial activity was obtained successfully and it had obvious inhibition effect on clinical common multidrug-resistant strains.

Immune peptides modelling of Culex pipiens sp by in silico methods.

Background: In the past 60 years, antibiotics have been critical in the fight against infectious diseases caused by bacteria and other microbes. Development of resistance to the antibiotics is emerging as a major public health issue which has resulted in the search for new antibiotics in order to maintain a pool of effective drugs at all times. Currently, there is a great interest in cationic peptides as antibiotics. These are reported to destroy the host cell membrane rather interacting with the other cell components, which may not face emergence of resistance. In mosquitoes, peptides like cecropin, defensin and gambicin reported to have inhibitory effect on bacteria, fungi and parasites. These peptides are well-characterized at both the biochemical and molecular level from Anopheles and Culex species, yet their 3D structures were not reported. Methods: Defensin, cecropin and gambicin immune peptides of Culex pipiens was characterised to have antiparasitic, antibacterial and antifungal activities. Since the crystal structure of defensin, cecropin and gambicin are not yet available their 3D structures were determined using homology modeling and Rosetta fragment insertion methods and were validated. Results: Stereo chemical evaluation indicated that defensin and gambicin showed that 100% residues of constructed model lie in the most favoured and allowed regions. Cecropin iso-forms A and B showed 100% while C showed 97.6% residues that lie in most favoured and allowed regions, which indicated quality models. Conclusion: Predicted model provide insight into their structure and aid in the development of novel antibiotic peptides.

Intein-mediated expression of cecropin in escherichia coli

Different strategies have been used to overcome the difficulties to produce antimicrobial peptides. Here we used Intein Mediated Purification with an Affinity Chitin-binding Tag (IMPACT-System, New England Biolabs) for the expression of the antimicrobial peptide cecropin to reduce its sensitivity to intracellular proteases and use its inducible self-cleaving capability to remove the carrier. Cecropin was cloned into suitable expression vector pTYB11, and expression induced by IPTG in Escherichia coli ER2566. The use of 22ºC induction allowed the expression of cecropin with its intein carrier in soluble form. Cell extracts were purified by chitin affinity chromatography and intein-mediated splicing of the target protein was achieved by thiol addition, obtaining a final yield of 2.5 mg cecropin/l. Cecropin cleaved from the intein had its proper biologically active form, showing a micromolar antimicrobial activity against Vibrio ordalii, Vibrio alginolyticus and Escherichia coli.

Cloning of the antibacterial peptide cecropin gene of Musca domestica larvae and its fusion expression in Escherichia coli / 中国人兽共患病学报

In the present study, the total RNA was extracted from three instar larvae of Musca domestica, the cDNA sequence encoding the ORF of cecropins was amplified by RT-PCR, and the target fragment was further sequenced after being cloned into T vector pUCm-T. Then, the cDNA sequence of the mature cecropins was amplified by PCR with recombinant plasmid pUCm-T/cecropin as template, the N-terminal rare codon GGA of E. coli was changed to the favorable codon GGC,and a Asn codon AAC was added in front of the stop coden TAA in the C- terminus. This mutant gene designated as mCecropin was then ligated with the fusion expression vector pGEX-4T-1. After restriction analysis and DNA sequencing, the positive recombinant plasmid pGEX-4T-1/mCecropin was transformed to different strains of E. coli cells and the fusion protein was expressed after IPTG induction. The fusion protein was assayed by SDS-PAGE and the E. coli BL21(DE3) cell was chosen as the host cell for the expression of the fusion protein. The expressed fusion protein GST-mCecropin was purified by GSTrap affinity coloum and the GST marker was then cleaved by thrombin. In this way, the fusion protein mCecropin with antibacterial activity was obtained after purification with HiTrap benzamidine column.

Antibacterial Cecropin and Its Application on Transgenic Plants for Bacterium Resistance / 中国生物工程杂志

Cecropin is a kind of heat-durable and broad-spectrum antibacterial polypeptides which has strong effect against bacteria,fungi,virus and some pathogenic microorganisms.Today Cecropin has been widely applied into plant genetic engineering,antiviral study,and inhibiting tumor cell proliferation.Its Structure-function relationship,antibacterial mechanism,and the application on transgenic plants for bacterium resistance was reviewed.Expression of Cecropin in plants has a great application potential in bacterium resistance.Deep analyses and research of molecular structure and action mechanism can promote the transgenic plants antibacterial research.

In vitro effects of cecropin-like antibacterial peptide from Helicobacter pylori on gastric cancer cells and gastric epithelial cells / 中华消化杂志

Objective To observe the in vitro effects of cecropin like anti bacterial peptide (2 20) from Helicobacter pylori ( H.pylori ) on gastric epithelial cells and gastric cancer cells. Methods The inhibition zone assay was used to determine anti bacterial activity and lethal concentrations of H.pylori anti bacterial peptide (2 20) and cecropin B on E.coli K12D31 (standard strain). The cytotoxicities and IC 50 of the H.pylori anti bacterial peptide (2 20) and cecropin B on human gastric epithelial cells and on gastric cancer cell lines were measured by MTT colorimatric methods. Results In vitro, the lethal concentrations (LC) of H.pylori anti bacterial peptide (2 20) and cecropin B on E.coli K12D31 were 65 ?mol/L and 0.44 ?mol respectively. The killing effects of cecropin B were more prominent than those of H.pylori anti bacterial peptide (2 20). The synthetic peptide H.pylori anti bacterial peptide(2 20) and cecropin B showed lytic or toxic activity against the human gastric cancer cell line SGC 7901 in a concentration dependent manner. The H.pylori anti bacterial peptide (2 20) stimulates the gastric epithelial cell GES 1 proliferation at the concentration ranging from 50 ?mol/L to 200 ?mol/L. The cecropin B has no lytic or toxic activety against gastric epithelial cell GES 1 at the concentration ranging from 5 ?mol/L to 20 ?mol/L. The IC 50 of H.pylori anti bacterial peptide (2 20)were 630 ?mol/L for GES 1 cells and 500 ?mol/L for SGC 7901 cells. The IC 50 of cecropin B were 75 ?mol/L for GES 1 cells and 25 ?mol/L for SGC 7901 cells respectively.Conclusions The H.pylori anti bacterial peptide (2 20) has a lower anti bacterial activity than cecropin B and there is a concentration dependent cytotoxic effect on the gastric cancer cells. The cecropin like antibacterial peptide from H.pylori anti bacterial peptide (2 20) stimulates the gastric epithelial cell proliferation at lower concentration and thus may be expected to increase the risk of gastric cancer.