Toll9 fromBombyx morifunctions as a pattern recognition receptor that shares features with Toll-like receptor 4 from mammals

Abstract

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.

Molecular mechanism underlying the TLR4 antagonistic and antiseptic activities of papiliocin, an insect innate immune response molecule

“…In contrast, in B. mori , LPS from gram-negative infection can activate the expression of AMPs such as cecropin B, whereas in mammals, TLR4 requires MD-2 for LPS recognition in response to gram-negative infections ( 20 , 27 ). Recently, B. mori Toll9 was found to colocalize with BmMD-2A and BmMD-2B on the surface of B. mori immune tissues ( 21 ). Simulated structures of B. mori MD-2A–lipid A and ML-2B–lipid A complexes based on the structure of the human MD2–lipid A complex revealed that similar to human MD-2, BmMD-2A and BmMD-2B contain two antiparallel β-sheets and a deep hydrophobic cavity.…”

“…The hydrophobic acyl chains of lipid A can bind to the human MD-2/LPS complex in this cavity, albeit in different directions, implying that B. mori MD-2–like proteins are the accessary proteins required for LPS recognition ( 20 ). Similar to mammalian TLR4 ectodomains, the homology-modeled structure of B. mori Toll9 using human TLR4 as a template contains a cysteine cluster carboxyl terminus to the leucine-rich repeats, with a characteristic horseshoe-like shape, suggesting that insect Toll9 evolutionarily clusters with mammalian TLR4 ( 21 ). The LPS recognition and signaling functions of the Toll9/MD-2–like protein complex of B. mori may be similar to that of the mammalian TLR4/MD-2 complex ( 21 ).…”

“…Insect Toll is highly similar to mammalian TLR, and Toll/TLRs are considered key regulators of the innate immune system in both insects and mammals ( 19 ). A recent study showed that in the silkworm, Bombyx mori , Toll9 recognizes LPS by interacting with two MD-2–related lipid recognition domains, named Toll9/MD-2A or Toll9/MD-2B, indicating their functional and evolutionary similarity with mammalian TLR4/MD-2 proteins ( 20 , 21 ). Insect Toll9 may be a pattern-recognition immune receptor similar to mammalian TLR4 in complex with MD-2 during LPS recognition and signaling ( 21 ).…”

“…A recent study showed that in the silkworm, Bombyx mori , Toll9 recognizes LPS by interacting with two MD-2–related lipid recognition domains, named Toll9/MD-2A or Toll9/MD-2B, indicating their functional and evolutionary similarity with mammalian TLR4/MD-2 proteins ( 20 , 21 ). Insect Toll9 may be a pattern-recognition immune receptor similar to mammalian TLR4 in complex with MD-2 during LPS recognition and signaling ( 21 ). Cecropins are a group of widely studied AMPs that play important roles in the innate immune response of insects ( 22 – 24 ).…”

“…Since then, several cecropin-like peptides have been identified in insects such as B. mori ( 26 ) and D. melanogaster ( 24 ). In B. mori, LPS can activate the expression of AMPs such as cecropin B, moricin, lebocin3, and attacin1 ( 21 , 27 ). Some insect cecropins have shown potent antibacterial activities and in vivo antiseptic activities, confirming their therapeutic potential ( 23 , 28 – 31 ).…”