Abstract
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. TmSpz-like comprised a cystine knot domain with six conserved cysteine residues that formed three disulfide bonds. Additionally, TmSpz-like exhibited the highest amino acid sequence similarity with T. castaneum Spätzle (TcSpz). In the phylogenetic tree, TmSpz-like and TcSpz 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.
Highlights
As invertebrates lack adaptive immunity, their immune responses against pathogens solely depend on innate immunity and physical barriers, such as chitinous shells
The findings of this study indicated that the viability of TmSpz-like knockdown larvae infected with E. coli was correlated with the levels of antimicrobial peptide (AMP)-encoding transcripts
TmSpz-like protein includes a signal peptide region at the N-terminus and a cysteine knot region in the C-terminus containing conserved cysteine residues that can potentially bind to the Toll receptor (Figure 1)
Summary
As invertebrates lack adaptive immunity, their immune responses against pathogens solely depend on innate immunity and physical barriers, such as chitinous shells. AMP production, which is the most conserved immune effector mechanism in invertebrates, is induced through the activation of the Toll and IMD signaling cascades. The Toll-like receptor (TLR)-nuclear factor kappa B (NF-κB) pathway has been elucidated in Drosophila In this signaling pathway, the PRRs recognize PAMPs, such as lipopolysaccharides (LPS), peptidoglycans (PGN), and β-glucans, on the surface of bacteria and fungi and trigger a multi-step proteolytic cascade of serine proteases. Modular serine protease (MSP), which activates the Toll ligand Spätzle (Spz) by processing the inactive pro-Spz, triggers the intracellular signaling cascade mechanism. Tenebrio GNBP1/PGRP-SA and GNBP3 bind to Lys-type PGN and β-1,3-glucans, respectively, of bacterial and fungal cell walls, and in the presence of Ca2+ ions, recruit pro-MSP and process it into activated MSP (aMSP). TmSpz proteins can function as central immune modulators for biosurfactant-mediated AMP production in T. molitor [5]
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