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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.

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.

SUBMITTER: Ko HJ

PROVIDER: S-EPMC8777057 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundThe excessive use of polystyrene as a packaging material has resulted in a rise in environmental pollution. Polystyrene waste has continually increased water pollution, soil pollution and the closing of landfill sites since it is durable and resistant to biodegradation. Therefore, the challenge in polystyrene disposal has caused researchers to look for urgent innovative and eco-friendly solutions for plastic degradation. The current study focuses on the isolation and identification of bacteria produced by the larvae of beetle Tenebrio molitor (yellow mealworms), that enable them to survive when fed with polystyrene foam as their sole carbon diet.Materials and methodsThe biodegradation of polystyrene by Tenebrio molitor was investigated by breeding and rearing the mealworms in the presence and absence of polystyrene. A comparison was made between those fed with a normal diet and those fed on polystyrene. The mealworms which were fed with polystyrene were then dissected and the guts were collected to isolate and identify the bacteria in their guts. The viability and metabolic activity of the isolates were investigated. The polymerase chain reaction (PCR) followed by sequencing was used for molecular identification of the isolates. The PCR products were directly sequenced using Sanger's method and the phylogenetic tree and molecular evolutionary analyses were constructed using MEGAX software with the Neighbour Joining algorithm. The evolutionary distances were computed using the Maximum Composite Likelihood method.ResultsThe decrease in mass of the polystyrene as feedstock confirmed that the mealworms were depending on polystyrene as their sole carbon diet. The frass egested by mealworms also confirmed the biodegradation of polystyrene as it contained very tiny residues of polystyrene. Three isolates were obtained from the mealworms guts, and all were found to be gram-negative. The sequencing results showed that the isolates were Klebsiella oxytoca ATCC 13182, Klebsiella oxytoca NBRC 102593 and Klebsiella oxytoca JCM 1665.ConclusionKlebsiella oxytoca ATCC 13182, Klebsiella oxytoca NBRC 102593 and Klebsiella oxytoca JCM 1665 maybe some of the bacteria responsible for polystyrene biodegradation.

Project description:Dorsal, a member of the nuclear factor-kappa B (NF-κB) family of transcription factors, is a critical downstream component of the Toll pathway that regulates the expression of antimicrobial peptides (AMPs) against pathogen invasion. In this study, the full-length ORF of Dorsal was identified from the RNA-seq database of the mealworm beetle Tenebrio molitor (TmDorX2). The ORF of TmDorX2 was 1,482 bp in length, encoding a polypeptide of 493 amino acid residues. TmDorX2 contains a conserved Rel homology domain (RHD) and an immunoglobulin-like, plexins, and transcription factors (IPT) domain. TmDorX2 mRNA was detected in all developmental stages, with the highest levels observed in 3-day-old adults. TmDorX2 transcripts were highly expressed in the adult Malpighian tubules (MT) and the larval fat body and MT tissues. After challenging the larvae with Staphylococcus aureus and Escherichia coli, the TmDorX2 mRNA levels were upregulated 6 and 9 h post infection in the whole body, fat body, and hemocytes. Upon Candida albicans challenge, the TmDorX2 mRNA expression were found highest at 9 h post-infection in the fat body. In addition, TmDorX2-knockdown larvae exposed to E. coli, S. aureus, or C. albicans challenge showed a significantly increased mortality rate. Furthermore, the expression of 11 AMP genes was downregulated in the gut and fat body of dsTmDorX2-injected larvae upon E. coli challenge. After C. albicans and S. aureus challenge of dsTmDorX2-injected larvae, the expression of 11 and 10 AMPs was downregulated in the gut and fat body, respectively. Intriguingly, the expression of antifungal transcripts TmTenecin-3 and TmThaumatin-like protein-1 and -2 was greatly decreased in TmDorX2-silenced larvae in response to C. albicans challenge, suggesting that TmDorX2 regulates antifungal AMPs in the gut in response to C. albicans infection. The AMP expression profiles in the fat body, hemocytes, gut, and MTs suggest that TmDorX2 might have an important role in promoting the survival of T. molitor larvae against all mentioned pathogens.

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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.

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