Tenebrio molitor Spätzle 1b Is Required to Confer Antibacterial Defense Against Gram-Negative Bacteria by Regulation of Antimicrobial Peptides

Abstract

Innate immunity is the ultimate line of defense against invading pathogens in insects. Unlike in the mammalian model, in the insect model, invading pathogens are recognized by extracellular receptors, which activate the Toll signaling pathway through an extracellular serine protease cascade. In the Toll-NF-κB pathway, the extracellular spätzle protein acts as a downstream ligand for Toll receptors in insects. In this study, we identified a novel Spätzle isoform (TmSpz1b) from RNA sequencing database of Tenebrio molitor. TmSpz1b was bioinformatically analyzed, and functionally characterized for the antimicrobial function by RNA interference (RNAi). The 702 bp open reading frame of TmSpz1b encoded a putative protein of 233 amino acid residues. A conserved cystine-knot domain with seven cysteine residues in TmSpz1b was involved in three disulfide bridges and the formation of a spätzle dimer. TmSpz1b was mostly expressed in the hemocytes of T. molitor late instar larvae. The mRNA expression of TmSpz1b was highly induced in the hemocytes after Escherichia coli, Staphylococcus aureus, and Candida albicans stimulation of T. molitor larvae. TmSpz1b silenced larvae were significantly more susceptible to E. coli infection. In addition, RNAi-based functional assay characterized TmSpz1b to be involved in the positive regulation of antimicrobial peptide genes in hemocytes and fat bodies. Further, the TmDorX2 transcripts were downregulated in TmSpz1b silenced individuals upon E. coli challenge suggesting the relationship to Toll signaling pathway. These results indicate that TmSpz1b is involved in the T. molitor innate immunity, causes the sequestration of Gram-negative bacteria by the regulatory action of antimicrobial peptides, and enhances the survival of T. molitor larvae.

Current Status of Immune Deficiency Pathway in Tenebrio molitor Innate Immunity

“…This protein is a zymogen and is cleaved to its mature form by a chain of serine protease activation, following pathogen recognition by PRRs (24,57). The immunological roles of Spz isoforms (Spz1b, Spz-like, Spz4, Spz5, and Spz6) in T. molitor have been investigated using RNAi (58)(59)(60)(61)(62). Among them, TmSpz1b, TmSpz-like, and TmSpz5 showed anti-Gramnegative bacterial (E. coli) activity (58,61,62).…”

“…The immunological roles of Spz isoforms (Spz1b, Spz-like, Spz4, Spz5, and Spz6) in T. molitor have been investigated using RNAi (58)(59)(60)(61)(62). Among them, TmSpz1b, TmSpz-like, and TmSpz5 showed anti-Gramnegative bacterial (E. coli) activity (58,61,62). silencing of TmSpz1b downregulated the expression of TmDorX1 and TmRel in the immune organs (58).…”

“…Among them, TmSpz1b, TmSpz-like, and TmSpz5 showed anti-Gramnegative bacterial (E. coli) activity (58,61,62). silencing of TmSpz1b downregulated the expression of TmDorX1 and TmRel in the immune organs (58). Similarly, TmSpz-like knockdown suppressed the expression of all NF-kB genes (61).…”

Emerging Evidence on Tenebrio molitor Immunity: A Focus on Gene Expression Involved in Microbial Infection for Host-Pathogen Interaction Studies

“…Another Spz isoform, Tm-Spz 1b (Genbank MZ708791), was identified and functionally characterised by Bae et al in 2021 [ 101 ]. Developmental and tissue-specific expression profiles established that Tm-Spz1b was mainly expressed in adult hemocytes and the late larval stage.…”

“…AMP-coding genes of RNAi larvae hemocytes and fat were downregulated in response to the E. coli challenge and Tm-DorX2 transcripts. Thus, these results suggested that Tm-Spz1b was required to confer antibacterial defence against Gram-negative bacteria but not against Gram-positive and fungal infections [ 101 ].…”