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The cystine knot protein Spätzle is a Toll receptor ligand that modulates the intracellular signaling cascade involved in the nuclear factor kappa B (NF-κB)-mediated regulation of antimicrobial peptide (AMP)-encoding genes. Spätzle-mediated activation of the Toll pathway is critical for the innate immune responses of insects against Gram-positive bacteria and fungi. In this study, the open reading frame (ORF) sequence of Spätzle-like from T. molitor ( TmSpz-like ) identified from the RNA sequencing dataset was cloned and sequenced. The 885-bp TmSpz-like ORF encoded a polypeptide of 294 amino acid residues. Tm Spz-like comprised a cystine knot domain with six conserved cysteine residues that formed three disulfide bonds. Additionally, Tm Spz-like exhibited the highest amino acid sequence similarity with T. castaneum Spätzle ( Tc Spz). In the phylogenetic tree, Tm Spz-like and Tc Spz were located within a single cluster. The expression of TmSpz-like was upregulated in the Malpighian tubules and gut tissues of T. molitor . Additionally, the expression of TmSpz-like in the whole body and gut of the larvae was upregulated at 24 h post- E. coli infection. The results of RNA interference experiments revealed that TmSpz-like is critical for the viability of E. coli -infected T. molitor larvae. Eleven AMP-encoding genes were downregulated in the E. coli -infected TmSpz-like knockdown larvae, which suggested that TmSpz-like positively regulated these genes. Additionally, the NF-κB-encoding genes ( TmDorX1 , TmDorX2 , and TmRelish ) were downregulated in the E. coli -infected TmSpz-like knockdown larvae. Thus, TmSpz-like plays a critical role in the regulation of AMP production in T. molitor in response to E. coli infection.

international journal of molecular sciences

TmSpz-like Plays a Fundamental Role in Response to E. coli but Not S. aureus or C. albican Infection in Tenebrio molitor via Regulation of Antimicrobial Peptide Production