TmIKKε Is Required to Confer Protection Against Gram-Negative Bacteria, E. coli by the Regulation of Antimicrobial Peptide Production in the Tenebrio molitor Fat Body.

Hye Jin Ko,Yong Seok Lee,Yong Hun Jo,Yeon Soo Han,Ki Beom Park,Ho Am Jang,Chang Eun Kim,Bharat Bhusan Patnaik,Snigdha Baliarsingh

doi:10.3389/fphys.2021.758862

Abstract

The inhibitor of nuclear factor-kappa B (NF-κB) kinase (IKK) is the core regulator of the NF-κB pathway against pathogenic invasion in vertebrates or invertebrates. IKKβ, -ε and -γ have pivotal roles in the Toll and immune deficiency (IMD) pathways. In this study, a homolog of IKKε (TmIKKε) was identified from Tenebrio molitor RNA sequence database and functionally characterized for its role in regulating immune signaling pathways in insects. The TmIKKε gene is characterized by two exons and one intron comprising an open reading frame (ORF) of 2,196 bp that putatively encodes a polypeptide of 731 amino acid residues. TmIKKε contains a serine/threonine protein kinases catalytic domain. Phylogenetic analysis established the close homology of TmIKKε to Tribolium castaneum IKKε (TcIKKε) and its proximity with other IKK-related kinases. The expression of TmIKKε mRNA was elevated in the gut, integument, and hemocytes of the last-instar larva and the fat body, Malpighian tubules, and testis of 5-day-old adults. TmIKKε expression was significantly induced by Escherichia coli, Staphylococcus aureus, and Candida albicans challenge in whole larvae and tissues, such as hemocytes, gut, and fat body. The knockdown of the TmIKKε messenger RNA (mRNA) expression significantly reduced the survival of the larvae against microbial challenges. Further, we investigated the induction patterns of 14 T. molitor antimicrobial peptides (AMPs) genes in TmIKKε gene-silencing model after microbial challenges. While in hemocytes, the transcriptional regulation of most AMPs was negatively regulated in the gut and fat body tissue of T. molitor, AMPs, such as TmTenecin 1, TmTenecin 4, TmDefensin, TmColeoptericin A, TmColeoptericin B, TmAttacin 1a, and TmAttacin 2, were positively regulated in TmIKKε-silenced individuals after microbial challenge. Collectively, the results implicate TmIKKε as an important factor in antimicrobial innate immune responses in T. molitor.

Highlights

The innate immune response represents the first line of defense in vertebrates and it is the only defense arsenal in invertebrates against microbial infections (Hoffmann et al, 1999)
The organization of the TmIKKε gene was deciphered from the T. molitor nucleotide database using the Tribolium castaneum IKKε (TcIKKε) amino acid sequence as a query in a tBLASTn analysis
Our research group has been successful in elucidating key genes of the Toll and immune deficiency (IMD) intracellular pathways, which are relevant in the context of humoral immunity in the coleopteran pest T. molitor (Patnaik et al, 2013, 2014; Tindwa et al, 2013)

Summary

Introduction

The innate immune response represents the first line of defense in vertebrates and it is the only defense arsenal in invertebrates against microbial infections (Hoffmann et al, 1999). Antimicrobial peptides (AMPs) production represents one of the crucial effector mechanisms of innate immunity in insects. Attacin B, which is one of two attacin genes identified from Hyphantria cunea, is strongly induced by gram-positive and gram-negative bacteria (Kwon et al, 2008). Another attacin gene identified from Spodoptera exigua has antimicrobial activity against Escherichia coli DH5α strain, Pseudomonas cichorii, Bacillus subtilis, and Listeria monocytogenes (Bang et al, 2012). In the Tenebrio model, in silico analysis and induction patterns of AMP genes that include tenecin-1 (defensin family), -2 (coleoptericin family), -3 (thaumatin-like protein family), and -4 (attacin family), thaumatin-like protein (TLP)-1, and -2, Attacin-1a, -1b, and 2, Defensin and Defensin-like, Coleoptericin-A, -B, and -C, and Cecropin-2 have been studied (Kim et al, 1998, 2017; Roh et al, 2009; Chae et al, 2012; Johnston et al, 2013; Zhu et al, 2014; Noh and Jo, 2016; Jo et al, 2018; Maistrou et al, 2018; Jang et al, 2020a,b; Ali Mohammadie Kojour et al, 2021)

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