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)

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)

Co-supplementation of dietary seaweed powder and antibacterial peptides improves broiler growth performance and immune function

The feasibility of Laminaria japonica powder (LJP) combined with cecropin as a dietary supplement to enhance broiler growth performance and immune function was evaluated in this study. In total, 648 one-day-old Arbor Acres broiler chicks were randomly distributed into nine numerically-equal treatment groups: T1 (control group; fed a basal diet); T2 (fed the basal diet supplemented with 1% LJP);T3 (fed the basal diet supplemented with 300mg cecropin/kg); and T4,T5,T6,T7,T8 and T9, individually fed with the dietary supplemented with varying levels of LJP and cecropin). Compared with the control, dietary of LJP or cecropin supplementation slightly improved feed conversion ratio (FCR). However, the dietary supplementation of LJP combined with cecropin significantly improved broiler growth performance during the periods of 1-21,21-42, and 1-42 days (p<0.05).The dietary supplementation of 3% LJP combined with 300 mg/kg cecropin significantly increased FCR, and serum Newcastle disease antibody titers and lymphocyte numbers during the periods of 1-21, 21-42, and 1-42 days (p<0.05). Cecal microorganisms were cultivated and the number of Escherichia coli and Lactobacillus colonies were counted. The dietary supplementation of LJP combined with cecropin remarkably inhibited E. coli growth and increased Lactobacillus growth. The results of this study demonstrate the feasibility of using LJP and cecropin as feed supplement for improving the growth performance and enhancing the immune function of broilers.(AU)

Co-supplementation of dietary seaweed powder and antibacterial peptides improves broiler growth performance and immune function

The feasibility of Laminaria japonica powder (LJP) combined with cecropin as a dietary supplement to enhance broiler growth performance and immune function was evaluated in this study. In total, 648 one-day-old Arbor Acres broiler chicks were randomly distributed into nine numerically-equal treatment groups: T1 (control group; fed a basal diet); T2 (fed the basal diet supplemented with 1% LJP);T3 (fed the basal diet supplemented with 300mg cecropin/kg); and T4,T5,T6,T7,T8 and T9, individually fed with the dietary supplemented with varying levels of LJP and cecropin). Compared with the control, dietary of LJP or cecropin supplementation slightly improved feed conversion ratio (FCR). However, the dietary supplementation of LJP combined with cecropin significantly improved broiler growth performance during the periods of 1-21,21-42, and 1-42 days (p<0.05).The dietary supplementation of 3% LJP combined with 300 mg/kg cecropin significantly increased FCR, and serum Newcastle disease antibody titers and lymphocyte numbers during the periods of 1-21, 21-42, and 1-42 days (p<0.05). Cecal microorganisms were cultivated and the number of Escherichia coli and Lactobacillus colonies were counted. The dietary supplementation of LJP combined with cecropin remarkably inhibited E. coli growth and increased Lactobacillus growth. The results of this study demonstrate the feasibility of using LJP and cecropin as feed supplement for improving the growth performance and enhancing the immune function of broilers.

A novel expression vector for the secretion of abaecin in Bacillus subtilis

ABSTRACT This study aimed to describe a Bacillus subtilis expression system based on genetically modified B. subtilis. Abaecin, an antimicrobial peptide obtained from Apis mellifera, can enhance the effect of pore-forming peptides from other species on the inhibition of bacterial growth. For the exogenous expression, the abaecin gene was fused with a tobacco etch virus protease cleavage site, a promoter Pglv, and a mature beta-glucanase signal peptide. Also, a B. subtilis expression system was constructed. The recombinant abaecin gene was expressed and purified as a recombinant protein in the culture supernatant. The purified abaecin did not inhibit the growth of Escherichia coli strain K88. Cecropin A and hymenoptaecin exhibited potent bactericidal activities at concentrations of 1 and 1.5 µM. Combinatorial assays revealed that cecropin A and hymenoptaecin had sublethal concentrations of 0.3 and 0.5 µM. This potentiating functional interaction represents a promising therapeutic strategy. It provides an opportunity to address the rising threat of multidrug-resistant pathogens that are recalcitrant to conventional antibiotics.(AU)