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)

Peptídeos antimicrobianos em Rhipicephalus (Boophilus) microplus / Antimicrobial peptides in Rhipicephalus (Boophilus) microplus

Background: The arthropod Rhipicephalus (Boophilus) microplus is a hematophagous ectoparasite that transmits a wide number of microorganisms to their host such as bacteria Anaplasma marginale. Anaplasmosis is responsible for serious damages to livestock due to mortality caused in herds, decrease in milk production and weight gain and expenses with prevention and control. Is an enzootic disease in temperate, subtropical and tropical countries. In these regions, cattle contamination may occur biologically by ticks, mechanically by flies or iatrogenically. The immune system of invertebrates has multiple mechanisms, but it is simpler than the immune system of vertebrates, however the ticks have a wide variety of protection mechanisms, including production of antimicrobial peptides (AMPs) which act directly against invading pathogens. To this date, only a few AMPs have been described in R. microplus, and little is known about the activity of these AMPs against A. marginale. Review: The tick R. microplus has several mechanisms to protect itself against invading microorganisms. Besides a protective cuticle and epithelia lining which are part of the first line of defense against pathogens, there are intermediate compounds of melanization, coagulation, phagocytosis, encapsulation, nodule formation, reactive oxygen species, proteins such as cystatins and additionally a vast repertoire of antimicrobial peptides (AMPs). However, the Anaplasma sp. have developed evolutionary mechanisms to be able to adapt and survive in this arthropod which is the main biological vector this pathogen. The AMPs can be expressed constitutively by the immune system, induced by infection, or by the recognition of surface components of microorganisms such as lipopolysaccharides (LPS) and peptidoglycan (PNG). However, through evolutionary events, the Anaplasma marginale lost genes encoding these components characteristic of the cell wall of Gram-negative bacteria, and thus, is likely that the major surface proteins (MSPs) are involved in its strengthening as the resistance to AMPs. Although the mechanisms of action of AMPs have not been fully elucidated, models are proposed to demonstrate how the interactions between lipid bilayer and AMP happen. More than 1,000 AMPs have been described in several groups of eukaryotes. In particular, amphibian peptides account for 592 of total AMPs representing a rich source of these molecules. Additionally, another 166 AMPs were isolated from insects. However, in R. microplus, few studies have described the existence of AMPs. The known R. microplus antimicrobial peptides are defensin and ixodidin (both isolated from hemocytes), the microplusin (isolated from female hemolymph and eggs), VTDCE ( isolated gut and ovary), and other two peptides characterized as fragments of bovine hemoglobin, Hb 33-91 and Hb 98-114, (isolated from engorged female gut). Conclusion: Since the silencing of genes encoding AMPs expressed in R. microplus decreases the number of A. marginale, it is suggested that this bacteria could adapt to support the tick immune defense mechanisms generating a symbiotic relationship, a evidence that the expression of AMPs can be manipulated by the pathogen to assist in its multiplication by a mechanism not yet defined, thus the Anaplasma sp. and the tick vector can live together allowing the bacteria transmission by the host. More studies about antimicrobial peptides expressed in R. microplus against invading microorganisms are necessary in order to improve the comprehension of its immune system and its competence for bovine anaplasmosis as a vector.