Identification and screening of potent antimicrobial peptides in arthropod genomes

Abstract

Using tBLASTn and BLASTp searches, we queried recently sequenced arthropod genomes and expressed sequence tags (ESTs) using a database of known arthropod cecropins, defensins, and attacins. We identified and synthesized 6 potential AMPs and screened them for antimicrobial activity. Using radial diffusion assays and microtiter antimicrobial assays, we assessed the in vitro antimicrobial effects of these peptides against several human pathogens including Gram-positive and Gram-negative bacteria and fungi. We also conducted hemolysis assays to examine the cytotoxicity of these peptides to mammalian cells. Four of the six peptides identified showed antimicrobial effects in these assays. We also created truncated versions of these four peptides to assay their antimicrobial activity. Two cecropins derived from the monarch butterfly genome (Danaus plexippus), DAN1 and DAN2, showed minimum inhibitory concentrations (MICs) in the range of 2–16 μg/ml when screened against Gram-negative bacteria. HOLO1 and LOUDEF1, two defensin-like peptides derived from red flour beetle (Tribolium castaneum) and human body louse (Pediculus humanus humanus), respectively, exhibited MICs in the range of 13–25 μg/mL against Gram-positive bacteria. Furthermore, HOLO1 showed an MIC less than 5μg/mL against the fungal species Candida albicans. These peptides exhibited no hemolytic activity at concentrations up to 200 μg/ml. The truncated peptides derived from DAN2 and HOLO1 showed very little antimicrobial activity. Our experiments show that the peptides DAN1, DAN2, HOLO1, and LOUDEF1 showed potent antimicrobial activity in vitro against common human pathogens, did not lyse mammalian red blood cells, and indicates their potential as templates for novel therapeutic agents against microbial infection.

Empirical Support for the Pattern of Competitive Exclusion between Insect Parasitic Fungi

“…The host immune modulation of parasite competition has been proposed in parasite–host interactive systems [ 51 , 52 ]. At least, for example, different numbers and families of antimicrobial peptide genes are encoded by different insects [ 53 ]. It was intriguing to find that the coinfection of ants or termites with M. anisopliae and the facultative saprophyte Aspergillus species largely resulted in the mycosis of insect cadavers by Aspergillus .…”

Cecropin-like antimicrobial peptide protects mice from lethal E.coli infection

“…We have found that DAN2 compromises the integrity of the bacterial membrane. Our in vitro study demonstrated that DAN2 does not lyse mammalian RBC which supports its potential use clinically [19]. Unlike bacterial cells which have 25% more anionic lipids that favor stronger electrostatic interaction, the mammalian cell membrane has large amounts of charge-neutral components, such as phosphatidylethanolamine, phosphatidylcholine, and sphingomyelin [29].…”

“…In our previous research, we have identified putative insect AMPs by searching newly sequenced arthropod genomes using known AMPs as homology templates [19]. We screened six cecropin and defensin derived peptides-DAN1, DAN2, HOLO1, LOUDEF1, INVICT1, and IXI for antimicrobial activity in vitro against several microbes including Gram-positive bacteria, Gram-negative bacteria and a single fungus.…”

New Insect Host Defense Peptides (HDP) From Dung Beetle (Coleoptera: Scarabaeidae) Transcriptomes

“…This strategy proved successful in this work, given that it identified 155 new putative HDP sequences from nine transcriptomes ( Supp Table S3 [online only ]). In contrast, an approach using similarity searches on deduced sequences derived genomic and expressed sequences tags (ESTs) from three insect species only identified six putative HDPs ( Duwadi et al 2018 ). One difference in these works is the presence of elements, such as introns and regulatory elements within the genomic sequences as they may limit the efficacy of gene identification by gene prediction methods.…”