Regulation of Antimicrobial Peptides in Aedes aegypti Aag2 Cells.

Rudian Zhang,Xiaojing Pang,Gong Cheng,Yibin Zhu,Renli Zhang,Xiaoping Xiao

doi:10.3389/fcimb.2017.00022

Abstract

Antimicrobial peptides (AMPs) are an important group of immune effectors that play a role in combating microbial infections in invertebrates. Most of the current information on the regulation of insect AMPs in microbial infection have been gained from Drosophila, and their regulation in other insects are still not completely understood. Here, we generated an AMP induction profile in response to infections with some Gram-negative, -positive bacteria, and fungi in Aedes aegypti embryonic Aag2 cells. Most of the AMP inductions caused by the gram-negative bacteria was controlled by the Immune deficiency (Imd) pathway; nonetheless, Gambicin, an AMP gene discovered only in mosquitoes, was combinatorially regulated by the Imd, Toll and JAK-STAT pathways in the Aag2 cells. Gambicin promoter analyses including specific sequence motif deletions implicated these three pathways in Gambicin activity, as shown by a luciferase assay. Moreover, the recognition between Rel1 (refer to Dif/Dorsal in Drosophila) and STAT and their regulatory sites at the Gambicin promoter site was validated by a super-shift electrophoretic mobility shift assay (EMSA). Our study provides information that increases our understanding of the regulation of AMPs in response to microbial infections in mosquitoes. And it is a new finding that the A. aegypti AMPs are mainly regulated Imd pathway only, which is quite different from the previous understanding obtained from Drosophila.

Highlights

Insects represent more than half of all known animals in the world and co-exist with numerous microorganisms in variable environments (Novotny et al, 2002; Engel and Grimaldi, 2004)
We examined the patterns of Antimicrobial peptides (AMPs) regulation in various microbe infections
Uninfected cells served as negative controls. 12 h later, the treated cells were collected to isolate total RNA for AMP detection by quantitative PCR. 3 Defensins (Def A, C and D), 6 Cecropins (Cec A, D, E, F, G, and N), and Gambicin were dramatically induced, while the other AMPs showed a modest induction or no response to the E. coli and S. marcescens infections (Figures 1A,B)

Summary

Introduction

Insects represent more than half of all known animals in the world and co-exist with numerous microorganisms in variable environments (Novotny et al, 2002; Engel and Grimaldi, 2004). Different insects express a variable spectrum of immune-induced AMPs. In Drosophila, 20 AMPs categorized into 7 groups, including 1 Defensin (Def ), 4 Cecropins (Cec), 2 Diptericins (Dpt), 4 Attacins (ATT), 1 Drosocin (Dro), 7 Drosomycins (Drs) and 1 Metchnikowin (Met), have been identified in the genome (Lemaitre and Hoffmann, 2007). In Drosophila, 20 AMPs categorized into 7 groups, including 1 Defensin (Def ), 4 Cecropins (Cec), 2 Diptericins (Dpt), 4 Attacins (ATT), 1 Drosocin (Dro), 7 Drosomycins (Drs) and 1 Metchnikowin (Met), have been identified in the genome (Lemaitre and Hoffmann, 2007) Parts of these genes clustered in a short area in Drosophila genome, and shared similar binding sites for regulatory factors so that they can AMP Regulation in Mosquito Cells be regulated in similar manners (Deng et al, 2009): Attacin, Diptericin, and Drosocin peptides effectively oppose Gramnegative bacterial infection (Wicker et al, 1990; Bulet et al, 1993; Asling et al, 1995). This large variation in the spectrum and constitution among insect AMPs indicates different regulatory patterns in response to microbial infections

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Results

Conclusion