Proteolytic activation and function of the cytokine Spatzle in the innate immune response of a lepidopteran insect, Manduca sexta

The innate immune response of insects includes induced expression of genes encoding a variety of antimicrobial peptides. The signaling pathways that stimulate this gene expression have been well characterized by genetic analysis in Drosophila melanogaster, but are not well understood in most other insect species. One such pathway involves proteolytic activation of a cytokine called Spatzle, which functions in dorsal-ventral patterning in early embryonic development and in the antimicrobial immune response in larvae and adults. We have investigated the function of Spatzle in a lepidopteran insect, Manduca sexta, in which hemolymph proteinases activated during immune responses have been characterized biochemically. Two cDNA isoforms for M. sexta Spatzle-1 differ because of alternative splicing, resulting in a 10 amino acid residue insertion in the pro-region of proSpatzle-1B that is not present in proSpatzle-1A. The proSpatzle-1A cDNA encodes a 32.7 kDa polypeptide that is 23% and 44% identical to D. melanogaster and Bombyx mori Spatzle-1, respectively. Recombinant proSpatzle-1A was a disulfide-linked homodimer. M. sexta hemolymph proteinase 8 cleaved proSpatzle-1A to release Spatzle-C108, a dimer of the C-terminal 108 residue cystine-knot domain. Injection of Spatzle-C108, but not proSpatzle-1A, into larvae stimulated expression of several antimicrobial peptides and proteins, including attacin-1, cecropin- 6, moricin, lysozyme, and the immunoglobulin domain protein hemolin, but did not significantly affect the expression of two bacteria-inducible pattern recognition proteins, immulectin-2 and b-1,3-glucan recognition protein-2. The results of this and other recent studies support a model for a pathway in which the clip-domain proteinase pro-hemolymph proteinase 6 becomes activated in plasma upon exposure to Gram-negative or Gram-positive bacteria or to b-1,3-glucan. Hemolymph proteinase 6 then activates pro-hemolymph proteinase 8, which in turn activates Spatzle-1. The resulting Spatzle-C108 dimer is likely to function as a ligand to activate a Toll pathway in M. sexta as a response to a wide variety of microbial challenges, stimulating a broad response to infection.