Manduca sexta moricin promoter elements can increase promoter activities of Drosophila melanogaster antimicrobial peptide genes

Abstract

Insects produce a variety of antimicrobial peptides (AMPs). Induction of insect AMP genes is regulated by the Toll and IMD (immune deficiency) pathways via NF-κB and GATA factors. Little is known about species-specific regulation of AMP genes. In this report, we showed that activities of most Manduca sexta and Drosophila melanogaster AMP gene promoters were regulated in a species-specific manner in Drosophila (Dipteran) S2 cells and Spodoptera frugiperda (Lepidopteran) Sf9 cells. A κB-GATA element (22bp) from M. sexta moricin (MsMoricin) promoter could significantly increase activities of Drosophila AMP gene promoters in S2 cells, and an MsMoricin promoter activating element (MPAE) (140bp) could increase activity of drosomycin promoter specifically in Sf9 cells. However, κB and GATA factors alone were not sufficient for MsMoricin gene activation, suggesting that other co-regulators may be required to fully activate AMP genes. Our results suggest that induction of insect AMP genes may require a transcription complex composed of common nuclear factors (such as NF-κB and GATA factors) and species-related co-regulators, and it is the co-regulators that may confer species-specific regulation of AMP genes. In addition, we showed that activity of Drosophila drosomycin promoter could be activated cooperatively by the inserted exogenous κB-GATA element and the endogenous κB element. These findings revealed an approach of engineering AMP genes with enhanced activities, which may lead to broad applications.

The immune signaling pathways of Manduca sexta

“…In contrast, Toll2 and 5 transcripts are highly abundant in fat body and their profiles of expression are closely similar to those of Dorsal, Serpent and Spätzle-1B. Interestingly, Manduca Dorsal and Serpent may interact with each other to activate moricin gene transcription (Rao et al, 2011). Toll2 and 5 containing a Cys-rich C-terminal domain, are more similar in domain structure to Drosophila Toll1.…”

Insect antimicrobial peptides and their applications

“…NF-κB and GATA binding sites have been identified in the promoter regions of several classes of AMP genes, including attacin genes (Sun et al, 1991; Taniai et al, 1996a; Taniai et al, 1996b) and moricin genes (Cheng et al, 2006). We have identified a 22-bp NF-κB-GATA cis -element, which can enhance the activity of D. melanogaster and M. sexta AMP gene promoters when inserted into the promoter regions (Rao et al, 2011). More importantly, we have identified a 140-bp cis -element, named m oricin p romoter a ctivating e lement (MPAE), which may contain binding sites for transcription factors specific for Lepidopteran insects, as insertion of MPAE into Drosophila drosomycin promoter can enhance the promoter activity specifically in S. frugiperda Sf9 cells (lepidopteran cell line) but not in Drosophila S2 cells (dipteran cell line) (Rao et al, 2011).…”

“…We have identified a 22-bp NF-κB-GATA cis -element, which can enhance the activity of D. melanogaster and M. sexta AMP gene promoters when inserted into the promoter regions (Rao et al, 2011). More importantly, we have identified a 140-bp cis -element, named m oricin p romoter a ctivating e lement (MPAE), which may contain binding sites for transcription factors specific for Lepidopteran insects, as insertion of MPAE into Drosophila drosomycin promoter can enhance the promoter activity specifically in S. frugiperda Sf9 cells (lepidopteran cell line) but not in Drosophila S2 cells (dipteran cell line) (Rao et al, 2011). Expression of moricin can be regulated by the Toll-Spätzle pathway (Zhong et al, 2012).…”

Identification of conserved and novel microRNAs in Manduca sexta and their possible roles in the expression regulation of immunity-related genes

“…However, there are no miRNA expression profiles presented and their abundances, based on the premise of expression co-regulation, were deduced from the microarray expression data of their adjacent genes. Differential regulation of D. melanogaster AMP genes in S2 and Sf 9 cell lines implied that intracellular immune signaling pathways involve species-specific regulators (Rao et al, 2011). Upon encountering Serratia marcescens or M. luteus , Apis mellifera workers mounted immune responses and, among the thirteen miRNAs predicted to regulate immunity in the honeybee, only two exhibited significant changes at 6 h after S. marcescens infection (Lourenco et al, 2013).…”