Toll9 from Bombyx mori functions as a pattern recognition receptor that shares features with Toll-like receptor 4 from mammals

Toll/Toll-like receptors (TLRs) are key regulators of the innate immune system in both invertebrates and vertebrates. However, while mammalian TLRs directly recognize pathogen-associated molecular patterns, the insect Toll pathway is thought to be primarily activated by binding Spätzle cytokines that are processed from inactive precursors in response to microbial infection. Phylogenetic and structural data generated in this study supported earlier results showing that Toll9 members differ from other insect Tolls by clustering with the mammalian TLR4 group, which recognizes lipopolysaccharide (LPS) through interaction with myeloid differentiation-2 (MD-2)–like proteins. Functional experiments showed that BmToll9 from the silkmoth Bombyx mori also recognized LPS through interaction with two MD-2–like proteins, previously named BmEsr16 and BmPP, that we refer to in this study as BmMD-2A and BmMD-2B, respectively. A chimeric BmToll9–TLR4 receptor consisting of the BmToll9 ectodomain and mouse TLR4 transmembrane and Toll/interleukin-1 (TIR) domains also activated LPS-induced release of inflammatory factors in murine cells but only in the presence of BmMD-2A or BmMD-2B. Overall, our results indicate that BmToll9 is a pattern recognition receptor for LPS that shares conserved features with the mammalian TLR4–MD-2–LPS pathway.

Loss of Cysteine Rich domain was critical for evolution of heterodimerization in Toll proteins

Toll proteins play roles in immunity/development which have largely remained conserved. However, there are differences in Toll biology as mammalian TLRs recognise pathogen associated molecular patterns (PAMPs) but not their invertebrate homologues. The reason for the same is not known. One critical molecular difference is absence of Cysteine Rich Domain (CRD) in vertebrate Tolls and their presence in invertebrates. Interestingly, in Drosophila, all Toll proteins have CRD except Toll9. This provided us the appropriate model to investigate significance of loss of CRD in Toll evolution. CRDs nudge protein dimerization by forming disulphide bonds hence we asked if they did same in Drosophila Toll-proteins. We tested if, Toll-1(which forms homodimer) can heterodimerize with Toll-9. We found that wildtype Toll-1 didn’t interact with Toll9 however; when CRD of Toll1 was deleted/mutated it formed heterodimer with Toll9. This indicates that presence of CRD limits Toll proteins to form homodimer and thus its loss was a critical event which pushed Toll proteins towards heterodimerization. We further show that Drosophila Toll9 can directly bind dsRNA, a PAMP. Interestingly, dsRNA affinity for toll-9 monomer was twice as that for the dimer, which can be attributed to CRD loss. Thus, we show that loss of CRD was a major step in Toll evolution as it resulted in functional diversity and was a first step towards heterodimer formation. Therefore, we propose that CRD loss was under positive selection and also that heterodimerization of Toll-proteins is an evolved property.One line summaryLoss of Cysteine Rich Domain in Drosophila Toll9 and recognition of dsRNA.

Toll and Toll-like receptors in Drosophila

The Drosophila Toll receptor controls the immune response to Gram-positive bacteria and fungi by activating a signalling pathway partially conserved throughout evolution. The Drosophila genome encodes eight additional Toll-related receptors, most of which appear to carry out developmental rather than immune functions. One exception may be Toll-9, which shares structural and functional similarities with mammalian TLRs.