Critical Roles of Spätzle5 in Antimicrobial Peptide Production Against Escherichia coli in Tenebrio molitor Malpighian Tubules

Abstract

The dimeric cytokine ligand Spätzle (Spz) is responsible for Toll pathway activation and antimicrobial peptide (AMP) production upon pathogen challenge in Tenebrio molitor. Here, we indicated that TmSpz5 has a functional role in response to bacterial infections. We showed that the highest expression of TmSpz5 is induced by Candida albicans. However, TmSpz5 knockdown reduced larval survival against Escherichia coli and Staphylococcus aureus. To evaluate the molecular mechanism underlying the observed survival differences, the role of TmSpz5 in AMP production was examined by RNA interference and microbial injection. T. molitor AMPs that are active against Gram-negative and -positive bacteria, including Tmtenecins, Tmattacins, Tmcoleoptericins, Tmtaumatin-like-proteins, and Tmcecropin-2, were significantly downregulated by TmSpz-5 RNAi in the Malpighian tubules (MTs) following a challenge with E. coli and S. aureus. However, upon infection with C. albicans the mRNA levels of most AMPs in the dsTmSpz5-injected group were similar to those in the control groups. Likewise, the expression of the transcription factors NF-κB, TmDorX2, and TmRelish were noticeably suppressed in the MTs of TmSpz5-silenced larvae. Moreover, E. coli-infected TmSpz5 knockdown larvae showed decreased antimicrobial activity in the MTs and hindgut compared with the control group. These results demonstrate that TmSpz5 has a defined role in T. molitor innate immunity by regulating AMP expression in MTs in response to E. coli.

Analysis of the Toll and Spaetzle Genes Involved in Toll Pathway-Dependent Antimicrobial Gene Induction in the Red Flour Beetle, Tribolium castaneum (Coleoptera; Tenebrionidae)

“…Complexes of spz and Toll proteins arrange hetero-trimers consisting of MyD88, Tube, and Pelle, which are intracellular components of the Toll pathway, leading to the expression of Toll-pathway-dependent AMPs (e.g., drosomycin) [ 10 , 11 , 23 , 24 ]. These involvements of Toll and spz in AMP production were reported in Bombyx mori (Lepidoptera; Bombycidae, Linnaeus, 1758) , Tenebrio molitor (Coleoptera; Tenebrionidae, Linnaeus, 1758), and Manduca sexta (Lepidoptera; Sphingidae, Linnaeus, 1763) [ 25 , 26 , 27 , 28 , 29 ].…”

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).…”

“…Similarly, TmSpz-like knockdown suppressed the expression of all NF-κB genes ( 61 ). The TmSpz5 -silenced larvae, however, showed a decreased expression of TmRel after Gram-negative bacterial infection but an increased expression of the same after a Gram-positive bacterial infection in the Malpighian tubules ( 62 ). Collectively, the activation of either Toll or Imd signaling pathways interferes with the other through unknown interactions between their components ( Table 3 ).…”

Editorial: Innate Immune System Guiding Physiological Plasticity in Invertebrates

“…The first article of this Research Topic ( Bae et al ) discusses the anti-Gram-negative function of another Toll ligand spätzle 1b (TmSpz1b) identified from the RNA sequencing database of T. molitor . Other studies, reported by the same research group, have critically characterized the function of T. molitor spätzle isoforms such as spätzle 5 (TmSpz5) and spätzle 6 (TmSpz6) in regulating AMP production in the Malpighian tubules in response to Escherichia coli , and against E. coli and Staphylococcus aureus , respectively ( Edosa et al, 2020 ; Ali Mohammadie Kojour et al, 2021 ). TmSpz1b was characterized as a secretory protein with a conserved cystine-knot domain as evidenced in other insects including spätzle protein in Drosophila melanogaster .…”