A Toll-Spätzle pathway in the tobacco hornworm, Manduca sexta

Abstract

Insects synthesize a battery of antimicrobial peptides (AMPs) and expression of AMP genes is regulated by the Toll and Imd (immune deficiency) pathways in Drosophila melanogaster. Drosophila Toll pathway is activated after Spätzle (Spz) is cleaved by Spätzle processing enzyme (SPE) to release the active C-terminal C106 domain (DmSpz-C106), which then binds to the Toll receptor to initiate the signaling pathway and regulate expression of AMP genes such as drosomycin. Toll and Spz genes have been identified in other insects, but interaction between Toll and Spz and direct evidence for a Toll-Spz pathway in other insect species have not been demonstrated. Our aim is to investigate a Toll-Spz pathway in Manduca sexta, and compare M. sexta and D. melanogaster Toll-Spz pathways. Co-immunoprecipitation (Co-IP) assays showed that MsTollecto (the ecto-domain of M. sexta Toll) could interact with MsSpz-C108 (the active C-terminal C108 domain of M. sexta Spz) but not with full-length MsSpz, and DmTollecto could interact with DmSpz-C106 but not DmSpz, suggesting that Toll receptor only binds to the active C-terminal domain of Spz. Co-expression of MsToll-MsSpz-C108, but not MsToll-MsSpz, could up-regulate expression of drosomycin gene in Drosophila S2 cells, indicating that MsToll-MsSpz-C108 complex can activate the Toll signaling pathway. In vivo assays showed that activation of AMP genes, including cecropin, attacin, moricin and lebocin, in M. sexta larvae by purified recombinant MsSpz-C108 could be blocked by pre-injection of antibody to MsToll, further confirming a Toll-Spz pathway in M. sexta, a lepidopteran insect.

The parasitic wasp Cotesia congregata uses multiple mechanisms to control host (Manduca sexta) behaviour

“…Increases in gene expression do not always lead to increased protein production (Zhang et al, 2014). Moreover, even if the proteins were produced, the pro-proteins need to be activated (Zhong et al, 2012). However, the dramatic drop in haemocyte number that occurs during wasp emergence supports the hypothesis that cytokine levels are high at this time (Adamo, 2005).…”

Insect antimicrobial peptides and their applications

“…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). …”

Reconfiguration of the immune system network during food limitation in the caterpillar Manduca sexta

“…We assessed the effects of food limitation on what appear to be key points in the immune system network of M. sexta, including: a pattern recognition molecule (hemolin), a cytokine that activates antimicrobial gene expression via the toll receptor (spätzle; An et al, 2010;Zhong et al, 2012), antimicrobial molecules (attacin and lysozyme), an antioxidant [glutathione (GSH)], and parts of the phenoloxidase (PO) cascade, including an activator [ prophenoloxidase activating proteinase-3 (PAP-3)] and an inhibitor of PAP-3, Serpin-3 (Fig. 1, see Jiang, 2008;Kanost and Gorman, 2008;Christen et al, 2012;Zhang et al, 2011;Cao et al, 2015;Chevignon et al, 2015).…”