93-kDa twin-domain serine protease inhibitor (Serpin) has a regulatory function on the beetle Toll proteolytic signaling cascade.
ABSTRACT Serpins are protease inhibitors that play essential roles in the down-regulation of extracellular proteolytic cascades. The core serpin domain is highly conserved, and typical serpins are encoded with a molecular size of 35-50 kDa. Here, we describe a novel 93-kDa protein that contains two complete, tandemly arrayed serpin domains. This twin serpin, SPN93, was isolated from the larval hemolymph of the large beetle Tenebrio molitor. The N-terminal serpin domain of SPN93 forms a covalent complex with the Spätzle-processing enzyme, a terminal serine protease of the Toll signaling cascade, whereas the C-terminal serpin domain of SPN93 forms complexes with a modular serine protease and the Spätzle-processing enzyme-activating enzyme, which are two different enzymes of the cascade. Consequently, SPN93 inhibited β-1,3-glucan-mediated Toll proteolytic cascade activation in an in vitro system. Site-specific proteolysis of SPN93 at the N-terminal serpin domain was observed after activation of the Toll proteolytic cascade in vivo, and down-regulation of SPN93 by RNAi sensitized β-1,3-glucan-mediated larval death. Therefore, SPN93 is the first serpin that contains twin tandemly arrayed and functionally active serpin domains that have a regulatory role in the larval Toll proteolytic signaling cascade.
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ABSTRACT: Understanding the evolutionary ecology of immune responses to persistent infection could provide fundamental insight into temporal dynamics or interactive mechanisms that could be co-opted for antibiotic treatment regimes. Additionally, identification of novel molecules involved in these processes could provide novel compounds for biotechnological development. The beetle Tenebrio molitor displays a high level of induced antimicrobial activity coincident with persistent immuno-resistant Staphylococcus aureus, and is the first invertebrate model for persistent infection. Here we present expressed sequence tags (ESTs) detected by suppression-subtraction hybridization of Tenebrio larvae after infection with S. aureus. Amongst others, we identified mRNAs coding for various oxidative enzymes and two antimicrobial peptides. These ESTs provide a foundation for mechanistic study of Tenebrio's immune system.Journal of insect physiology 10/2012; 58(12). DOI:10.1016/j.jinsphys.2012.09.009 · 2.50 Impact Factor
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ABSTRACT: microRNAs (miRNAs) play significant regulatory roles in gene expression at the post-transcriptional level. This includes modulating processes such as development, immunity, cancer, and host-pathogen interactions. It was recently shown that the phylogenetically deeply conserved miRNA, miR-8, plays a role in maintaining the homeostasis of immunity by suppressing the production of anti-microbial peptides. In this study, we show that miR-8 from the insect Plutella xylostella positively regulates the transcript levels of the serine protease inhibitor Serpin 27, which has been shown to regulate activation of the Toll pathway and prophenoloxidase involved in the melanization response in insects. Interestingly, miR-8 is downregulated following parasitization by Diadegma semiclausum leading to significant declines in Serpin 27 transcript levels. This allows upregulation of antimicrobial peptides, such as gloverin, that are controlled by the Toll pathway and activation of proteolytic cascades essential for humoral immune responses to foreign invasion.RNA biology 06/2013; 10(8). DOI:10.4161/rna.25481 · 5.38 Impact Factor
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ABSTRACT: Tenebrio molitor has been seriously investigated as a model insect in elucidating Toll signaling pathway and related regulators of innate immunity. However, little is known with regards to the genomic information in T. molitor. In an attempt towards exploiting the rich transcriptomics data that would offer further insights into the study on insect immunity through potential screening of immune-related genes in the model insect, we constructed a cDNA library (library titer = 5.0 × 105pfu/ml) of T. molitor larvae and analyzed expressed sequence tag (EST) sequences from 1056 clones. The base calling and quality check of obtained chromatogram files were performed by using the Phred program (trim_alt 0.05 (P-score > 20). After removal of vector and 100 bp and less sequences, 1023 sequences were generated having an average insert size of 792 bp, including 160 clusters, 164 contigs and 387 singletons through clustering and assembling process using the TGI Clustering Tools (TGICL) package. The unique EST sequences were searched against the NCBI nr database by local BLAST (blastx, E < e−5) with 940 sequences showing significant hits. Subsequently, KOG (clusters of orthologous groups for eukaryotic complete genomes) analysis was conducted (blastx, E < e−10) towards prediction of transcriptomal functions, leading to the categorization of 638 genes. The majority of genes belonged to Z category (cytoskeleton-related genes), R category (general function prediction), and C category (energy production and conversion related genes). These basic EST datasets and their bioinformatics analysis will be helpful in investigating the biological mechanism and molecular pathway related genes involved in innate immunity mechanisms of T. molitor.Entomological Research 01/2013; 43(3):162-170. DOI:10.1111/1748-5967.12019 · 0.33 Impact Factor