TmRelish is required for regulating the antimicrobial responses to Escherichia coli and Staphylococcus aureus in Tenebrio molitor

Abstract

Relish, a transcription factor, is a critical downstream component of the immune deficiency (Imd) pathway and regulates host defense against bacterial infection by mediating antimicrobial peptide (AMP) synthesis. Understanding the immunological function of the mealworm beetle, Tenebrio molitor Relish (TmRelish) will be instructive in understanding insect immunity. In the present study, full-length oRf of TmRelish was retrieved from T. molitor-expressed sequence tags and RNA-seq database. The predicted TmRelish amino acid sequence contained an N-terminal Rel-homology domain; an Ig-like, plexin, and transcription factor domain; ankyrin repeat motifs; a nuclear localization signal; and a C-terminal death domain and shared the highly conserved structure of the Relish proteins of other insect species. TmRelish mRNA was detected in all developmental stages of the insect; however, the highest levels were detected in the larval gut tissue and adult hemocytes. TmRelish mRNA level was upregulated in the fat body, hemocyte, and gut tissue 9 h after infection of T. molitor larvae by the gram-negative bacteria, Escherichia coli. Furthermore, TmRelish knockdown led to significantly higher mortality of the E. coli-infected larvae, and significantly lower mortality of larvae infected with Staphylococcus aureus or Candida albicans. To elucidate the possible cause of mortality, we measured AMP transcription in the fat body, hemocytes, gut, and Malpighian tubules (MTs) of T. molitor larvae. TmRelish knockdown suppressed the expression of nine AMP genes in the larval fat body and gut tissue during E. coli infection, suggesting that TmRelish positively regulates AMP expression in both immunerelated tissues, in response to E. coli challenge. Furthermore, negative regulation of some AMPs by TmRelish in the MTs, gut and hemocytes in response to C. albicans infection suggests a crosstalk between the Toll and Imd pathways.

Tenebrio molitor PGRP-LE Plays a Critical Role in Gut Antimicrobial Peptide Production in Response to Escherichia coli

“…In this manuscript, we report that silencing of TmPGRP-LE reduced the expression levels of larval gut TmRelish, TmDorX1, and TmDorX2 after E. coli challenge. This is consistent with our recent report demonstrating that all the aforementioned AMPs were positively regulated by TmDorX2 (Keshavarz et al, 2019) and TmRelish (Keshavarz et al, 2020). These results leave open the possibility that detection of invading E. coli by TmPGRP-LE in the gut of T. molitor larvae results in signal transduction to both TmDorX2 and TmRelish.…”

“…As opposed to Drosophila, S. aureus-infected T. molitor larvae exhibited increased mortality following TmPGRP-LE silencing, and the transcription of nine AMPs decreased in TmPGRP-LE knockdown larval gut. It should also be mentioned that TmRelish is a positive regulator of TmTene1, TmTene4, TmAtt1b, TmAtt2, TmCole1, and TmCole2 in the gut of S. aureus-infected larvae (Keshavarz et al, 2020) suggesting that the Imd pathway is critical for combatting infection of the larval gut infected with S. aureus via AMP production. We further show that the involvement of TmPGRP-LE in the control of the Imd pathway is limited to only some AMPs in the fat body and hemocytes of S. aureus-challenged larvae.…”

“…We observed a notable effect of TmPGRP-LE silencing on the mRNA expression of transcription proteins for both Imd (TmRelish) and Toll (TmDorX1 and 2) signaling pathways in response to C. albicans infection in the fat body and gut. In this context, following C. albicans challenge, downregulation of TmPGRP-LE-induced genes (i.e., AMPs) is mediated by TmDorX2 in the gut (Keshavarz et al, 2019), whereas the AMP transcription levels are mainly regulated by TmRelish in the fat body (Keshavarz et al, 2020).…”

Two Roles for the Tenebrio molitor Relish in the Regulation of Antimicrobial Peptides and Autophagy-Related Genes in Response to Listeria monocytogenes

“…In T. molitor fat body, the expression levels of 10 AMPs were upregulated in non-TmRelish-silenced groups after L. monocytogenes infection; however, RNAi TmRelish-injected larvae showed downregulated expression of TmTene1 and -4; TmDef1 and -2; TmCole1 and -2, TmAtt1a, -1b, and -2; and TmCec2. The results of the current study agree with the results of our previous studies on the gram-positive bacteria, S. aureus, in which all listed AMPs were found to be downregulated in the fat body of dsTmRelish-treated larvae following S. aureus challenge [32]. Therefore, TmRelish plays a critical role in response to Gram-positive bacteria, including intracellular bacteria in the larval fat body of T. molitor.…”

“…Previous studies of the immune function of TmRelish have shown that this transcription factor can be detected in all examined tissues, with high levels of expression in the gut, fat body, and hemocytes, and relatively low levels of expression in Malpighian tubules and integument [32]. Since the highest levels of TmRelish expression are in the most important immune-related tissues in T. molitor, these tissues were chosen to study the time-course of TmRelish expression using qRT-PCR.…”