Characterization of the immune induced antimicrobial peptide in Drosophila melanogaster and Drosophila ananassae

Abstract

Insects can recognize invading pathogens and initiate an immune response. Among them, Drosophila has emerged as an invertebrate model for investigating innate immune responses in which antimicrobial peptides play a crucial role. In the present study, immune-induced antimicrobial peptides were characterized in D. melanogaster and D. ananassae using the agar well diffusion method, HPLC, SDS-PAGE and LC-MS/MS after infection with either S. aureus or E. coli. The HPLC revealed two and three differentially induced components, respectively, in D. melanogaster and D. ananassae fl ies infected with S. aureus and E. coli. The tricine SDS-PAGE analysis also revealed two and fi ve differentially induced proteins, respectively, in D. melanogaster and D. ananassae infected with E. coli. In E. coli infected fl ies, the ~6 kDa band was produced at higher level. Based on LCMS/MS and Mascot analysis, the peptide was identifi ed as a putative cecropin A-like peptide, and the data suggested that both species of Drosophila have exhibited a clear immune response. The fl ies were also able to discriminate between bacteria, as this putative cecropin A-like peptide was produced in fl ies infected with E. coli but not S. aureus. * Corresponding author; e-mail: knagarajv@gmail.com INTRODUCTION The fruit fl y, Drosophila melanogaster has innate immunity against invading microbes. This includes both cellular and humoral immune responses (Lye, 2018; Meghashree & Nagaraj, 2020). Antimicrobial peptides (AMPs) are an important component in the fi rst line of defence (Yuchen et al., 2019). AMPs are endogenous peptides with a molecular weight (MW) of ~2–22 kDa and they are released by the fat body (analogue of the liver) into haemolymph to clear off the microbial infections (Troha et al., 2019). The interactions of AMPs with Gram-positive and Gramnegative bacteria differ. The positively charged AMPs selectively interact with prokaryotes having a negatively charged bacterial cell-wall, including lipopolysaccharides (LPS) and phospholipids. Based on the available data (FlyBase), nine distinct classes of AMPs (23 members) are identifi ed in Drosophila (Thurmond et al., 2019). Among them, attacin, diptericin, cecropin and drosocin are produced in response to Gram-negative bacterial infections (Imd pathway), metchnikowin and defensin in response to a Gram-positive bacterial infection (Toll pathway) and drosomycin only in response to fungal infection (Sheehan et al., 2018). As fruit fl ies are genetically similar in the way they combat diseases as humans, they can be used to evaluate miEur. J. Entomol. 118: 355–363, 2021 doi: 10.14411/eje.2021.037