Functional Divergence among Silkworm Antimicrobial Peptide Paralogs by the Activities of Recombinant Proteins and the Induced Expression Profiles

Wanying Yang,Bo Wang,Xiang Tan,Mingqiang Ye,Yang Cao,Yadong Huang,Xiaojuan Deng,Tingcai Cheng,Qingyou Xia,Zhonghuai Xiang,Huiyu Yi,Dong Han,Joy Sturtevant

doi:10.1371/journal.pone.0018109

Abstract

Antimicrobial peptides are small-molecule proteins that are usually encoded by multiple-gene families. They play crucial roles in the innate immune response, but reports on the functional divergence of antimicrobial peptide gene families are rare. In this study, 14 paralogs of antimicrobial peptides belonging to cecropin, moricin and gloverin families were recombinantly expressed in pET expression systems. By antimicrobial activity tests, peptides representing paralogs in the same family of cecropin and moricin families, displayed remarkable differences against 10 tested bacteria. The evolutionary rates were relatively fast in the two families, which presented obvious functional divergence among paralogs of each family. Four peptides of gloverin family had similar antimicrobial spectrum and activity against tested bacteria. The gloverin family showed similar antimicrobial function and slow evolutionary rates. By induced transcriptional activity, genes encoding active antimicrobial peptides were upregulated at obviously different levels when silkworm pupae were infected by three types of microbes. Association analysis of antimicrobial activities and induced transcriptional activities indicated that the antimicrobial activities might be positively correlated with induced transcriptional activities in the cecropin and moricin families. These results suggest that representative BmcecB6, BmcecD and Bmmor as the major effector genes have broad antimicrobial spectrum, strong antimicrobial activity and high microbe-induced expression among each family and maybe play crucial roles in eliminating microbial infection.

Highlights

In insects, innate immunity is the first line of defense against invading microbes, and antimicrobial peptides (AMPs) play crucial roles in killing invaders and preventing infection
By testing the antimicrobial activity of recombinant proteins and induced expression profile in Drosophila adults, we found the functional divergence of drosomycin family and explored the coevolutionary mechanism in insect immune defense
By in vitro testing the antimicrobial activity of recombinant AMPs and by Real-time RT-PCR testing induced expression profiles of these AMP genes after infection of day-2 pupae with three types of microbes, our results indicated that these paralogs among either cecropin family or moricin family have obviously diversified in antimicrobial function and show a correlation between broad antimicrobial spectrum and strong antibacterial activity with high induced expression level of AMP genes, similar to drosomycin family in Drosophila

Summary

Introduction

Innate immunity is the first line of defense against invading microbes, and antimicrobial peptides (AMPs) play crucial roles in killing invaders and preventing infection. They have a low molecular weight, high heat stability, a broad spectrum, and high antimicrobial activity. Most AMPs are encoded by multiple gene families, as seen in the completed genomic sequences of several insects, such as the cecropin and drosomycin families in Drosophila melanogaster [1], the cecropin, moricin and gloverin families in Bombyx mori [2,3]. AMPs have extensive gene duplication and rapid gene turnover, but positive selected sites are absent [4]. Both rapid gene duplication and positive selection have been found in AMPs from frogs [16,17]

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