A Spatzle/Toll pathway in Manduca sexta

Insects produce a variety of antimicrobial peptides (AMPs). Expression of AMP genes is induced by microbial components and regulated by the Toll and Imd (immune deficiency) pathways. In Drosophila melanogaster, the Toll pathway can be activated by Gram positive bacteria and fungi, resulting in activation of Spätzle processing enzyme (SPE) that cleaves Spätzle (Spz) to its active form (carboxyl-terminal C106 fragment). Active Spz-C106 then binds to the Toll receptor, initiating the assembly of the Toll induced signaling complex (TISC), which then regulates expression of some AMPs, such as Drosomycin, Defensin and Metchnikowin. Toll receptors and Spätzle proteins have also been identified in other insect species, including the tobacco hornworm, Manduca sexta. However, other than in D. melanogaster, the Toll-Spätzle pathway has not been confirmed in other insects. Our aim is to investigate whether M. sexta has a similar Toll-Spätzle pathway. S2 cell lines expressing M. sexta Toll (MsToll), Spätzle (MsSpz) and the active form of MsSpätzle (MsSpz-C108) were established. The Co-immunoprecipitation (CoIP) assays showed that MsToll^ecto(the extracellular domain of MsToll) could interact with MsSpz-C108 but not with MsSpz. By co-expressing MsToll with MsSpz or MsSpz-C108 in S2 cells, we showed that MsSpz-C108, but not MsSpz, could activate Drosomycin gene in S2 cells, indicating that a Toll-Spätzle pathway is present in M. sexta.