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)

The effect of dietary cecropin AD on intestinal health, immune response and disease resistance of juvenile turbot (Scophthalmus maximus L.).

Cecropin AD (CAD) is a commercial cationic antimicrobial peptide that has been seldom studied in marine fish. This study investigated the effects of dietary CAD on intestinal health, immune response, disease resistance, and growth performance of turbot. A diet using fishmeal and plant protein as the main protein resources was used as the control (crude protein 53%, crude lipid 12%). CAD was supplemented into the control diet at the level of 250, 500, 750, and 1000 mg kg-1 to formulate four experimental diets, C1, C2, C3, and C4, respectively. No significant difference was observed in fish growth performance, feed utilization efficiency and whole-body composition among all groups. Dietary CAD significantly increased the activity of lysozyme and complement component 3 level in both serum and distal intestine (DI), as well as the immunoglobulin M content in DI. The gene expression of immune cytokines such as IFN-γ, IL-1ß, and chemokine SmCCL19, and the goblet cell number in DI were also significantly increased by dietary CAD supplementation. Compared with the control group, the microbiota analysis indicated group C4 showed significantly decreased α-diversity, obvious alternation in dominant bacteria composition at phylum level, different clustering, and significantly decreased relative abundance of Lactobacillus. Besides, the relative abundance of Bacteroides was significantly decreased in groups C1, C3, and C4. In addition, the lowest mortality of turbot challenged with Edwardsiella tarda was observed in fish fed diets C2 and C3. In conclusion, moderate levels of CAD in diet of turbot improved the intestinal immune response without disrupting the intestinal bacterial community, and enhanced the disease resistance. However, dietary CAD at 1000 mg kg-1 greatly affected the intestinal bacterial composition and showed potentially inhibitory effects towards Lactobacillus.

Anti-inflammatory activities of Aedes aegypti cecropins and their protection against murine endotoxin shock.

BACKGROUND: Mosquitoes are armed with physiologically active compounds to suppress the host immunity including host inflammatory reaction. However, the specific anti-inflammatory components in mosquitoes remain unknown. RESULTS: By searching for the immunomodulatory molecules from the mosquito Aedes aegypti (Diptera: Culicidae) at NCBI for anti-inflammatory function, five cecropins (for short in this study: AeaeCec1, 2, 3, 4 and 5) were selected. AeaeCec1-5 efficiently inhibited the expression of inducible nitric oxide synthase (iNOS), nitrite, tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages and human peripheral blood mononuclear cells (PBMCs) with low toxicity to mammalian cells. Among the five analogues, AeaeCec5 had the strongest anti-inflammatory activity, and generated an additive effect with other AeaeCec peptides. In a mouse model of endotoxin shock, AeaeCec1-5 effectively reduced TNF-α, IL-1ß and IL-6 expression in lungs, serum and peritoneal lavage and correspondingly reduced lung damage and edema, with AeaeCec5 showing the best protection. In mice infected with Escherichia coli or Pseudomonas aeruginosa, administration of AeaeCec5 reduced the production of TNF-α, IL-1ß and IL-6 and correspondingly reduced lung tissue damage. These effects of Ae. aegypti AeaeCec1-5 were attributed to an efficient inhibition of the activation of mitogen-activated protein kinases (MAPKs) and transcriptional nuclear factor-κB (NF-κB) signaling pathways, as well as partial neutralization of LPS. CONCLUSIONS: The current work characterized the specific anti-inflammatory agents in Ae. aegypti and provided AeaeCec5 as a potent anti-endotoxin peptide that could serve as the basis for the development of anti-inflammatory therapy.

Antitumor effects of cecropin B-LHRH' on drug-resistant ovarian and endometrial cancer cells.

BACKGROUND: Luteinizing hormone-releasing hormone receptor (LHRHr) represents a promising therapeutic target for treating sex hormone-dependent tumors. We coupled cecropin B, an antimicrobial peptide, to LHRH', a form of LHRH modified at carboxyl-terminal residues 4-10, which binds to LHRHr without interfering with luteinizing hormone (LH) and follicle-stimulating hormone (FSH) secretion. This study aimed to assess the antitumor effects of cecropin B-LHRH' (CB-LHRH') in drug-resistant ovarian and endometrial cancers. METHODS: To evaluate the antitumor effects of CB-LHRH', three drug resistant ovarian cancer cell lines (SKOV-3, ES-2, NIH:OVCAR-3) and an endometrial cancer cell line (HEC-1A) were treated with CB-LHRH'. Cell morphology changes were assessed using inverted and electron microscopes. In addition, cell growth and cell cytotoxicity were measured by MTT assay and LDH release, respectively. In addition, hemolysis was measured. Furthermore, radioligand receptor binding, hypersensitization and minimal inhibitory concentrations (against Staphylococcus aureus, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Pseudomonas aeruginosa, and Acinetobacter baumannii) were determined. Finally, the impact on tumor growth in BALB/c-nu mice was assessed in an ES-2 xenograft model. RESULTS: CB-LHRH' bound LHRHr with high-affinity (dissociation constant, Kd = 0.252 ± 0.061 nM). Interestingly, CB-LHRH' significantly inhibited the cell viability of SKOV-3, ES-2, NIH:OVCAR-3 and HEC-1A, but not that of normal eukaryotic cells. CB-LHRH' was active against bacteria at micromolar concentrations, and caused no hypersensitivity in guinea pigs. Furthermore, CB-LHRH' inhibited tumor growth with a 23.8 and 20.4 % reduction in tumor weight at 50 and 25 mg/kg.d, respectively. CONCLUSIONS: CB-LHRH' is a candidate for targeted chemotherapy against ovarian and endometrial cancers.

Cecropin-P17, an analog of Cecropin B, inhibits human hepatocellular carcinoma cell HepG-2 proliferation via regulation of ROS, Caspase, Bax, and Bcl-2.

Cecropin-P17 is a peptide derived from Cecropin B. In this study, we investigated the effects and relative mechanisms of Cecropin-P17 in a human liver cancer cell line (HepG-2) in vitro and in vivo. A cell viability assay, Annexin V/propidium iodide assay, western blot, flow cytometry, quantitative real-time polymerase chain reaction, and a tumor-xenograft model were applied to elucidate the mechanism exerted by Cecropin-P17 on HepG-2 cells. Cecropin-P17 significantly inhibited the proliferation of HepG-2 cells and demonstrated low cytotoxicity to normal liver cells in vitro. The apoptotic rate of HepG-2 cells was increased after Cecropin-P17 treatment together with increased production of reactive oxygen species. Moreover, Cecropin-P17 stimulated caspase-3, caspase-9, and Bax and inhibited Bcl-2 on both the transcriptional and translational levels. Finally, Cecropin-P17 significantly suppressed tumor growth in a HepG-2-bearing nude mouse model. All of these results indicated that Cecropin-P17 could be a potential agent for the treatment of liver cancer.