IKKß regulates antimicrobial innate immune responses in the yellow mealworm, Tenebrio molitor.

Toll and IMD pathways regulate antimicrobial innate immune responses in insect model systems. The transcriptional activation of antimicrobial peptides (AMPs) confers humoral immunity in the host against invaded pathogens. The IKK kinase complex (IKKα, IKKß, and the regulatory subunit IKKγ/NEMO) centrally regulates the NF-κB response to various stimuli. It triggers an appropriate antimicrobial immune response in the host. In this study, a TmIKKß (or TmIrd5) homolog was screened from the RNA-seq database of the coleopteran beetle, Tenebrio molitor. A single exon characterizes the TmIKKß gene, and the open reading frame (ORF) comprises of 2112 bp that putatively encodes a polypeptide of 703 amino acid residues. TmIKKß contains a serine/threonine kinase domain and is phylogenetically close to Tribolium castaneum IKKß homolog (TcIKKß). TmIKKß transcripts were highly expressed in the early pupal (P1) and adult (A5) stages. Among the tissues, TmIKKß showed higher expression in the integument of the last instar larvae and the fat body and hemocytes of 5-day-old adults. TmIKKß mRNA was upregulated post-E. coli challenge to the host. Moreover, RNAi-based TmIKKß mRNA silencing increased host larvae' susceptibility against E. coli, S. aureus and C. albicans. TmIKKß RNAi in the fat body led to a downregulation in mRNA expression of ten out of fourteen AMP genes, including TmTenecin1, -2, and -4; TmDefensin, and -like; TmColeoptericinA, and -B; and TmAttacin1a, -1b, and -2, suggesting the requirement of the gene in antimicrobial innate immune responses. Further, a decrease in the mRNA expression of NF-κB factors such as TmRelish, TmDorsal1, and TmDorsal2 in the fat body of T. molitor larvae was observed post-microorganisms challenge. Thus, TmIKKß regulates antimicrobial innate immune responses in T. molitor.

Characterization of a Lipopolysaccharide- and Beta-1,3-Glucan Binding Protein (LGBP) from the Hepatopancreas of Freshwater Prawn, Macrobrachium rosenbergii, Possessing Lectin-Like Activity.

The study focuses on the isolation, characterization, and expression analysis of a lectin from the hepatopancreas of Macrobrachium rosenbergii. The protein was isolated by affinity chromatography on a melibiose-agarose column. The molecular weight of the native protein was found to be ~120 kDa which consists of a single polypeptide of ~39.5 kDa. On mass spectrometric analysis, the protein was identified as lipopolysaccharide- and beta-1,3-glucan binding protein (LGBP). LGBP showed hemagglutination with rabbit RBC like a lectin and its carbohydrate-binding specificity was determined by the hemagglutination inhibition test. The protein also showed antibacterial activity against two Gram-negative bacteria Vibrio harveyi and Aeromonas sobria, and one Gram positive bacteria Bacillus cereus in the disc diffusion test. Rabbit antiserum was raised against the purified LGBP and used to develop a sandwich ELISA system for quantitation of the protein in hepatopancreas and serum samples of M. rosenbergii. The expression of the LGBP transcripts in muscle, hepatopancreas, and gill tissues from M. rosenbergii juveniles at 72 h post-challenge of V. harveyi was not modulated as noticed in qPCR analysis. However, significant increases in the concentrations of LGBP protein in hepatopancreas (5.23 ± 0.45 against 3.43 ± 0.43 mg/g tissue in control) and serum (1.08 ± 0.14 against 0.61 ± 0.08 µg/ml in control) were observed in the challenged group of prawns in ELISA suggesting its putative role against bacterial infections. The study for the first time characterized the native LGBP of M. rosenbergii showing a multifunctional role in immunity.

Molecular cloning, sequence characterization, and expression analysis of C-type lectin (CTL) and ER-Golgi intermediate compartment 53-kDa protein (ERGIC-53) homologs from the freshwater prawn, Macrobrachium rosenbergii.

Lectin protein families are diverse and multi-functional in crustaceans. The carbohydrate-binding domains (CRDs) of lectins recognize the molecular patterns associated with pathogens and orchestrate important roles in crustacean defense. In this study, two lectin homologs, a single CRD containing C-type lectin (CTL) and an L-type lectin (LTL) domain containing endoplasmic reticulum Golgi intermediate compartment 53 kDa protein (ERGIC-53) were identified from the freshwater prawn, Macrobrachium rosenbergii. The open reading frames of MrCTL and MrERGIC-53 were 654 and 1,515 bp, encoding polypeptides of 217 and 504 amino acids, respectively. Further, MrCTL showed a 20-amino acid transmembrane helix region and 10 carbohydrate-binding residues within the CRD. MrERGIC-53 showed a signal peptide region, a type-I transmembrane region, and a coiled-coil region at the C-terminus. Phylogenetic analysis revealed a close relationship between MrCTL and MrLectin and M. nipponense CTL (MnCTL), whereas MrERGIC-53 shared high sequence identity with Eriocheir sinensis ERGIC-53 and Penaeus vannamei MBL-1. A homology-based model predicted small carbohydrate-combining sites with a metal-binding site for ligand binding (Ca2+ binding site) in MrCTL and beta-sheets connected by short loops and beta-bends forming a dome-shaped beta-barrel structure representing the LTL domain of MrERGIC-53. Quantitative real-time polymerase chain reaction detected MrCTL and MrERGIC-53 transcripts in all examined tissues, with particularly high levels observed in hemocytes, hepatopancreas, and mucosal-associated tissues, such as the stomach and intestine. Further, the expression levels of MrCTL and MrERGIC-53 transcripts were remarkably altered after V. harveyi challenge, suggesting putative function in host innate immunity. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10499-022-00845-3.

Tenebrio molitor Spätzle 1b Is Required to Confer Antibacterial Defense Against Gram-Negative Bacteria by Regulation of Antimicrobial Peptides.

Innate immunity is the ultimate line of defense against invading pathogens in insects. Unlike in the mammalian model, in the insect model, invading pathogens are recognized by extracellular receptors, which activate the Toll signaling pathway through an extracellular serine protease cascade. In the Toll-NF-κB pathway, the extracellular spätzle protein acts as a downstream ligand for Toll receptors in insects. In this study, we identified a novel Spätzle isoform (TmSpz1b) from RNA sequencing database of Tenebrio molitor. TmSpz1b was bioinformatically analyzed, and functionally characterized for the antimicrobial function by RNA interference (RNAi). The 702 bp open reading frame of TmSpz1b encoded a putative protein of 233 amino acid residues. A conserved cystine-knot domain with seven cysteine residues in TmSpz1b was involved in three disulfide bridges and the formation of a spätzle dimer. TmSpz1b was mostly expressed in the hemocytes of T. molitor late instar larvae. The mRNA expression of TmSpz1b was highly induced in the hemocytes after Escherichia coli, Staphylococcus aureus, and Candida albicans stimulation of T. molitor larvae. TmSpz1b silenced larvae were significantly more susceptible to E. coli infection. In addition, RNAi-based functional assay characterized TmSpz1b to be involved in the positive regulation of antimicrobial peptide genes in hemocytes and fat bodies. Further, the TmDorX2 transcripts were downregulated in TmSpz1b silenced individuals upon E. coli challenge suggesting the relationship to Toll signaling pathway. These results indicate that TmSpz1b is involved in the T. molitor innate immunity, causes the sequestration of Gram-negative bacteria by the regulatory action of antimicrobial peptides, and enhances the survival of T. molitor larvae.

TmSpz-like Plays a Fundamental Role in Response to E. coli but Not S. aureus or C. albican Infection in Tenebrio molitor via Regulation of Antimicrobial Peptide Production.

The cystine knot protein Spätzle is a Toll receptor ligand that modulates the intracellular signaling cascade involved in the nuclear factor kappa B (NF-κB)-mediated regulation of antimicrobial peptide (AMP)-encoding genes. Spätzle-mediated activation of the Toll pathway is critical for the innate immune responses of insects against Gram-positive bacteria and fungi. In this study, the open reading frame (ORF) sequence of Spätzle-like from T. molitor (TmSpz-like) identified from the RNA sequencing dataset was cloned and sequenced. The 885-bp TmSpz-like ORF encoded a polypeptide of 294 amino acid residues. TmSpz-like comprised a cystine knot domain with six conserved cysteine residues that formed three disulfide bonds. Additionally, TmSpz-like exhibited the highest amino acid sequence similarity with T. castaneum Spätzle (TcSpz). In the phylogenetic tree, TmSpz-like and TcSpz were located within a single cluster. The expression of TmSpz-like was upregulated in the Malpighian tubules and gut tissues of T. molitor. Additionally, the expression of TmSpz-like in the whole body and gut of the larvae was upregulated at 24 h post-E. coli infection. The results of RNA interference experiments revealed that TmSpz-like is critical for the viability of E. coli-infected T. molitor larvae. Eleven AMP-encoding genes were downregulated in the E. coli-infected TmSpz-like knockdown larvae, which suggested that TmSpz-like positively regulated these genes. Additionally, the NF-κB-encoding genes (TmDorX1, TmDorX2, and TmRelish) were downregulated in the E. coli-infected TmSpz-like knockdown larvae. Thus, TmSpz-like plays a critical role in the regulation of AMP production in T. molitor in response to E. coli infection.

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.

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. IKKß, -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.

IKKγ/NEMO Is Required to Confer Antimicrobial Innate Immune Responses in the Yellow Mealworm, Tenebrio Molitor.

IKKγ/NEMO is the regulatory subunit of the IκB kinase (IKK) complex, which regulates the NF-κB signaling pathway. Within the IKK complex, IKKγ/NEMO is the non-catalytic subunit, whereas IKKα and IKKß are the structurally related catalytic subunits. In this study, TmIKKγ was screened from the Tenebrio molitor RNA-Seq database and functionally characterized using RNAi screening for its role in regulating T. molitor antimicrobial peptide (AMP) genes after microbial challenges. The TmIKKγ transcript is 1521 bp that putatively encodes a polypeptide of 506 amino acid residues. TmIKKγ contains a NF-κB essential modulator (NEMO) and a leucine zipper domain of coiled coil region 2 (LZCC2). A phylogenetic analysis confirmed its homology to the red flour beetle, Tribolium castaneum IKKγ (TcIKKγ). The expression of TmIKKγ mRNA showed that it might function in diverse tissues of the insect, with a higher expression in the hemocytes and the fat body of the late-instar larvae. TmIKKγ mRNA expression was induced by Escherichia coli, Staphylococcus aureus, and Candida albicans challenges in the whole larvae and in tissues such as the hemocytes, gut and fat body. The knockdown of TmIKKγ mRNA significantly reduced the survival of the larvae after microbial challenges. Furthermore, we investigated the tissue-specific induction patterns of fourteen T. molitor AMP genes in TmIKKγ mRNA-silenced individuals after microbial challenges. In general, the mRNA expression of TmTenecin1, -2, and -4; TmDefensin1 and -2; TmColeoptericin1 and 2; and TmAttacin1a, 1b, and 2 were found to be downregulated in the hemocytes, gut, and fat body tissues in the TmIKKγ-silenced individuals after microbial challenges. Under similar conditions, TmRelish (NF-κB transcription factor) mRNA was also found to be downregulated. Thus, TmIKKγ is an important factor in the antimicrobial innate immune response of T. molitor.

Lectin-Like Activity of Hemocyanin in Freshwater Prawn, Macrobrachium rosenbergii.

Lectins are proteins that bind to the carbohydrate moieties on surface of bacteria, erythrocytes and other cells of invertebrates causing agglutination and mediate in recognition of foreign substances. In the present study, we isolated and characterized a lectin molecule present in the hemolymph of Macrobrachium rosenbergii, an important cultured freshwater prawn. Lectin in serum samples of adult prawns was assessed through hemagglutination (HA) test using rabbit RBC that showed a titre ranging from 16 to 64. This serum hemagglutinin was confirmed as a C-type lectin based on its dependency on calcium ions towards binding to rabbit RBCs. The hemagglutinin was also found to be stable at the pH range of 5.0-10.0 and temperature range of 10-40 °C. Of various sugars and glycoproteins tested in hemagglutination inhibition assay, the serum lectin was found specific only to N-acetylneuraminic acid and fetuin with respective minimum inhibitory concentrations at 50 mM and 0.31 mg/ml. Further, the lectin was purified by affinity chromatography on rabbit erythrocyte stroma, which showed hemagglutination with rabbit RBC. In electrophoretic analyses, the purified lectin showed one band with molecular weight of ~ 427 kDa in native gradient PAGE, and its two constituent polypeptide chains of ~ 81 and ~ 73 kDa in SDS-PAGE. These polypeptides were analysed in MALDI-TOF/TOF mass spectrometry and identified as hemocyanins. It was hence, concluded that hemocyanin in M. rosenbergii possesses lectin-like activity.

Aedes albopictus Autophagy-Related Gene 8 (AaAtg8) Is Required to Confer Anti-Bacterial Gut Immunity.

Autophagy is an important process by which pathogens and damaged or unused organelles are eliminated. The role of autophagy in development and the immune response to pathogens is well established. Autophagy-related protein 8 (Atg8) is involved in the formation of the autophagosome and, with the help of the serine protease Atg4, mediates the delivery of both vesicles and the autophagosome to the vacuole. Here, we cloned the Aedes albopictus autophagy-related protein 8 (AaAtg8) gene and characterized its role in the innate immunity of the mosquito against microbial infections. AaAtg8 is comprised of an open reading frame (ORF) region of 357 bp encoding a polypeptide of 118 amino acid residues. A domain analysis of AaAtg8 revealed an Atg8 ubiquitin-like domain, Atg7/Atg4 interaction sites, and peptide binding sites. The AaAtg8 mRNA expression was high in the Malpighian tubules and heads of both sugar-fed and blood-fed adult female mosquitoes. The expression level of AaAtg8 mRNA increased in the midgut and abdominal carcass following being challenged with Listeria monocytogenes. To investigate the role of AaAtg8 in the innate immune responses of Ae. albopictus, AaAtg8 gene-silenced adult mosquitoes were challenged by injection or by being fed microorganisms in blood. High mortality rates were observed in mosquitoes in which AaAtg8 was silenced after challenges of microorganisms to the host by blood feeding. This suggests that Atg8-autophagy plays a critical role in the gut immunity in Ae. albopictus.

Molecular Cloning and Expression Analysis of Three Suppressors of Cytokine Signaling Genes (SOCS5, SOCS6, SOCS7) in the Mealworm Beetle Tenebrio molitor.

Suppressors of cytokine signaling (SOCS) influence cytokine and growth factor signaling by negatively regulating the Janus kinase (JAK)-signal transducers and activators of transcription (STAT) pathway to maintain homeostasis during immune responses. However, functional characterization of SOCS family members in invertebrates is limited. Here, we identified and evaluated three SOCS genes (type I sub-family) in the mealworm beetle Tenebrio molitor. The full-length open reading frames (ORFs) of TmSOCS5, TmSOCS6, and TmSOCS7 comprised of 1389, 897, and 1458 nucleotides, encoding polypeptides of 462, 297, and 485 amino acids, respectively. The SH2 and SOCS box domains of the TmSOCS C-terminal region were highly conserved. Phylogenetic analysis revealed that these SOCS genes were clustered within the type I subfamily that exhibits the highest amino acid identity with Tribolium castaneum SOCS genes. Contrary to TmSOCS7 expression, the expression levels of TmSOCS5 and TmSOCS6 were lower in the larval, pupal, and adult stages. In larvae and adults, the expression levels of TmSOCS5 and TmSOCS6 were highest in the hemocytes and ovaries, respectively. SOCS transcripts were also highly upregulated in the hemocytes of T. molitor larvae within 3⁻6 h post-infection with the fungus Candida albicans. Collectively, these results provide valuable information regarding the involvement of TmSOCS type-I subfamily in the host immune response of insects.

Transcriptome analysis of the threatened snail Ellobium chinense reveals candidate genes for adaptation and identifies SSRs for conservation genetics.

Ellobium chinense (Pfeiffer, 1854) is a brackish pulmonate species that inhabits the bases of mangrove trees and is most commonly found in salt grass meadows. Threats to mangrove ecosystems due to habitat degradation and overexploitation have threatened the species with extinction. In South Korea, E. chinense has been assessed as vulnerable, but there are limited data on its population structure and distribution. The nucleotide and protein sequences for this species are not available in databases, which limits the understanding of adaptation-related traits. We sequenced an E. chinense cDNA library using the Illumina platform, and the subsequent bioinformatics analysis yielded 227,032 unigenes. Of these unigenes, 69,088 were annotated to matched protein and nucleotide sequences in databases, for an annotation rate of 30.42%. Among the predominant gene ontology terms, cellular and metabolic processes (under the biological process category), membrane and cell (under the cellular component category), and binding and catalytic activity (under the molecular function category) were noteworthy. In addition, 4850 unigenes were distributed to 15 Kyoto Encyclopaedia of Genes and Genomes based enrichment categories. Among the candidate genes related to adaptation, angiotensin I converting enzyme, adenylate cyclase activating polypeptide, and AMP-activated protein kinase were the most prominent. A total of 15,952 simple sequence repeats (SSRs) were identified in sequences of > 1 kb in length. The di- and trinucleotide repeat motifs were the most common. Among the repeat motif types, AG/CT, AC/GT, and AAC/GTT dominated. Our study provides the first comprehensive genomics dataset for E. chinense, which favors conservation programs for the restoration of the species and provides sufficient evidence for genetic variability among the wild populations.

Immunomodulatory Effects of Extracellular ß-Glucan Isolated from the King Oyster Mushroom Pleurotus eryngii (Agaricomycetes) and Its Sulfated Form on Signaling Molecules Involved in Innate Immunity.

The aim of this study was to determine, using murine RAW 264.7 macrophages, the immunomodulatory effect of extracellular ß-glucan isolated from Pleurotus eryngii (PEBG) and its sulfated derivative (PEBG-S) on signaling molecules implicated in host innate immunity. ß-Glucan was extracted and purified from the mycelial culture using optimal medium concentrations. It was then chemically converted to its sulfated form. Monosaccharide composition of ß-glucan was characterized with p-aminobenzoic acid ethyl ester-derivatized sugars through highperformance liquid chromatography analysis. Fourier transform infrared structural analysis showed an S=O bond at 1250 cm-1 and C-S-O binding at 815 cm-1 in PEBG-S. 13C nuclear magnetic resonance analysis showed 1,3-linked α-D-mannopyranosyl and 1,3-ß-D-glucopyranosyl in PEBG-S. A concentration-dependent increase of nitric oxide production was noticed in RAW 264.7 cells treated with PEBG-S or PEBG; those treated with PEBG-S showed less cytotoxicity than those treated with PEBG. Cellular levels of tumor necrosis factor-α, interleukin-1ß, and interleukin-6 were increased by PEBG and PEBG-S treatment, suggesting that they have immunomodulatory activity. Real-time polymerase chain reaction array revealed that the expression levels of nuclear factor-κB and Toll-like receptor signaling genes in cells were upregulated by PEBG and PEBG-S. Moreover, the expression of the ß-glucan receptor dectin-2 was significantly upregulated by PEBG and PEBG-S treatment, reflecting immune activation through the dectin-2-Syk-(CARD9/Bcl-10/MALT1) pathway. Our results suggest that PEBG-S could be used as an effective adjuvant or immune enhancer that can be sustainably produced by recycling the by-product of mycelial culture.

The Silencing of a 14-3-3ɛ Homolog in Tenebrio molitor Leads to Increased Antimicrobial Activity in Hemocyte and Reduces Larval Survivability.

The 14-3-3 family of phosphorylated serine-binding proteins acts as signaling molecules in biological processes such as metabolism, division, differentiation, autophagy, and apoptosis. Herein, we report the requirement of 14-3-3ɛ isoform from Tenebrio molitor (Tm14-3-3ɛ) in the hemocyte antimicrobial activity. The Tm14-3-3ɛ transcript is 771 nucleotides in length and encodes a polypeptide of 256 amino acid residues. The protein has the typical 14-3-3 domain, the nuclear export signal (NES) sequence, and the peptide binding residues. The Tm14-3-3ɛ transcript shows a significant three-fold expression in the hemocyte of T. molitor larvae when infected with Escherichia coli Tm14-3-3ɛ silenced larvae show significantly lower survival rates when infected with E. coli. Under Tm14-3-3ɛ silenced condition, a strong antimicrobial activity is elicited in the hemocyte of the host inoculated with E. coli. This suggests impaired secretion of antimicrobial peptides (AMP) into the hemolymph. Furthermore, a reduction in AMP secretion under Tm14-3-3ɛ silenced condition would be responsible for loss in the capacity to kill bacteria and might explain the reduced survivability of the larvae upon E. coli challenge. This shows that Tm14-3-3ɛ is required to maintain innate immunity in T. molitor by enabling antimicrobial secretion into the hemolymph and explains the functional specialization of the isoform.

Mutan: A mixed linkage α-[(1,3)- and (1,6)]-d-glucan from Streptococcus mutans, that induces osteoclast differentiation and promotes alveolar bone loss.

Mutan is an extracellular polysaccharide of Streptococcus mutans (S. mutans) that consists of α-(1,3)-linked glucose residues in main chains and α-(1,6) bonds in side chains. In the present study, mutan was isolated from S. mutans, and its structural characteristics were determined using Fourier-transform infrared spectroscopy (FT-IR) and (13)C nuclear magnetic resonance (NMR) spectroscopy. The effects of mutan on RANKL-induced osteoclast differentiation in RAW 264.7 cells were examined. Furthermore, microCT and morphometric analyses were used to determine the contribution of mutan to alveolar bone loss in the maxilla of a rat periodontitis model. Mutan increased (more than 2-fold) RANKL-induced osteoclast differentiation in a dose-dependent manner. Mutan also enhanced the alveolar bone loss in the rat maxilla 2.3-fold. In mutan-treated rats, the bone mineral density, bone volume, trabecular number, and trabecular thickness decreased, whereas trabecular separation significantly increased. In addition, mutan and lipopolysaccharide (LPS) induced similar microarray profiles in RAW 264.7 cells. A total of 43 genes related to osteoclastogenesis were differentially expressed after either mutan or LPS treatment. Five-fold increases in the expression of several genes, including IL-1ß, IL-1α, IL-6, and chemokine ligands, were observed in mutan-treated RAW 264.7 cells. These results suggest a molecular mechanism for the inflammation induced by S. mutans during the establishment of periodontal disease.

Brazilin isolated from Caesalpinia sappan L. inhibits rheumatoid arthritis activity in a type-II collagen induced arthritis mouse model.

BACKGROUND: Caesalpinia sappan L. extracts exhibit great therapeutic potential, and have been shown to have analgesic and anti-inflammatory properties. This study aimed to understand the anti-rheumatoid activity of brazilin that was isolated from ethyl acetate extract of C. sappan L. The evaluations were conducted in mice with type-II collagen-induced arthritis (CIA). METHODS: Brazilin was purified via preparative HPLC and identified by mass spectrometry and 1H/13C NMR analysis. DBA/1J mice were divided into four groups (n=10). Three groups of mice received intradermal injections of inducer bovine type-II collagen (BTIIC; 2 mg/ml in 0.05 ml acetic acid) and 0.1 ml of booster complete Freund's adjuvant (CFA). A second injection of BTIIC with booster incomplete Freund's adjuvant (ICFA) was given subsequently after 21 days. On 22nd day, purified brazilin (10 mg/kg body weight) or the disease-modifying anti-rheumatic drug methotrexate (3 mg/kg body weight) was administered intraperitoneally daily or every three days for 21 days, respectively to two groups of mice. At the 42nd day, mice sera were collected, and the levels of pro-inflammatory cytokines and stress enzyme markers in serum were measured using standard immunoassay methods. The microstructure and morphometric analyses of the bones were assessed using high-resolution microfocal computed tomography. RESULTS: Brazilin isolated from C. sappan reduced the arthritis index score and the extent of acute inflammatory paw edema in CIA-mice. The bone mineral density was significantly (p<0.05) lower in only-CIA mice, and appeared to increase commensurate with methotrexate and brazilin administration. Brazilin prevented joint destruction, surface erosion, and enhanced bone formation as revealed by microstructural examinations. Brazilin markedly attenuated mouse CIA and reduced the serum levels of inflammatory cytokines including TNF-α, IL-1ß, and IL-6. CONCLUSIONS: Brazilin purified from C. sappan L. shows protective efficacy in CIA mouse, and may be useful to treat chronic inflammatory disorders including rheumatoid arthritis.

Depletion of autophagy-related genes ATG3 and ATG5 in Tenebrio molitor leads to decreased survivability against an intracellular pathogen, Listeria monocytogenes.

Macroautophagy (autophagy) is an evolutionarily conserved catabolic process involved in physiological and developmental processes including cell survival, death, and innate immunity. Homologues of most of 36 originally discovered autophagy-related (ATG) genes in yeast have been characterized in higher eukaryotes including insects. In this study, the homologues of ATG3 (TmATG3) and ATG5 (TmATG5) were isolated from the coleopteran beetle, Tenebrio molitor by expressed sequence tag and RNAseq approaches. The cDNA of TmATG3 and TmATG5 comprise open-reading frame sizes of 963 and 792 bp encoding polypeptides of 320 and 263 amino acid residues, respectively. TmATG3 and TmATG5 mRNA are expressed in all developmental stages, and mainly in fat body and hemocytes of larvae. TmATG3 and TmATG5 showed an overall sequence identity of 58-95% to other insect Atg proteins. There exist clear one-to-one orthologs of TmATG3 and TmATG5 in Tribolium and that they clustered together in the gene tree. Depletion of TmATG3 and TmATG5 by RNA interference led to a significant reduction in survival ability of T. molitor larvae against an intracellular pathogen, Listeria monocytogenes. Six days post-Listeria challenge, the survival rate in the dsEGFP-injected (where EGFP is enhanced green fluorescent protein) control larvae was significantly higher (55%) compared to 4 and 3% for TmATG3 and TmATG5 double-stranded RNA injected larvae, respectively. These data suggested that TmATG3 and TmATG5 may play putative role in mediating autophagy-based clearance of Listeria in T. molitor model.

Cloning, characterization and effect of TmPGRP-LE gene silencing on survival of Tenebrio molitor against Listeria monocytogenes infection.

Peptidoglycan recognition proteins (PGRPs) are a family of innate immune molecules that recognize bacterial peptidoglycan. PGRP-LE, a member of the PGRP family, selectively binds to diaminopimelic acid (DAP)-type peptidoglycan to activate both the immune deficiency (Imd) and proPhenoloxidase (proPO) pathways in insects. A PGRP-LE-dependent induction of autophagy to control Listeria monocytogenes has also been reported. We identified and partially characterized a novel PGRP-LE homologue, from Tenebrio molitor and analyzed its functional role in the survival of the insect against infection by a DAP-type PGN containing intracellular pathogen, L. monocytogenes. The cDNA is comprised of an open reading frame (ORF) of 990 bp and encodes a polypeptide of 329 residues. TmPGRP-LE contains one PGRP domain, but lacks critical residues for amidase activity. Quantitative RT-PCR analysis showed a broad constitutive expression of the transcript at various stages of development spanning from larva to adult. RNAi mediated knockdown of the transcripts, followed by a challenge with L. monocytogenes, showed a significant reduction in survival rate of the larvae, suggesting a putative role of TmPGRP-LE in sensing and control of L. monocytogenes infection in T. molitor. These results implicate PGRP-LE as a defense protein necessary for survival of T. molitor against infection by L. monocytogenes.

Molecular cloning, sequence characterization and expression analysis of a CD63 homologue from the coleopteran beetle, Tenebrio molitor.

CD63, a member of the tetraspanin membrane protein family, plays a pivotal role in cell growth, motility, signal transduction, host-pathogen interactions and cancer. In this work, the cDNA encoding CD63 homologue (TmCD63) was cloned from larvae of a coleopteran beetle, Tenebrio molitor. The cDNA is comprised of an open reading frame of 705 bp, encoding putative protein of 235 amino acid residues. In silico analysis shows that the protein has four putative transmembrane domains and one large extracellular loop. The characteristic "Cys-Cys-Gly" motif and "Cys188" residues are highly conserved in the large extracellular loop. Phylogenetic analysis of TmCD63 revealed that they belong to the insect cluster with 50%-56% identity. Analysis of spatial expression patterns demonstrated that TmCD63 mRNA is mainly expressed in gut and Malphigian tubules of larvae and the testis of the adult. Developmental expression patterns of CD63 mRNA showed that TmCD63 transcripts are detected in late larval, pupal and adult stages. Interestingly, TmCD63 transcripts are upregulated to the maximum level of 4.5 fold, in response to DAP-type peptidoglycan during the first 6 h, although other immune elicitors also caused significant increase to the transcript level at later time-points. These results suggest that CD63 might contribute to T. molitor immune response against various microbial pathogens.

Molecular and immunohistochemical characterization of granulin gene encoded in Pieris rapae granulovirus genome.

Pieris rapae granulovirus (PiraGV) is highly pathogenic to the cabbage butterfly (P. rapae), an important pest of cultivated cabbages and mustard crops. It therefore holds significant promise towards exploitation as a potent bio-control agent in the field controlling the pest population. Whole-genome elucidation of the Korean isolate of the granulovirus (PiraGV-K), reported the presence of a granulin gene corresponding to ORF 1 in its genome. Comprehensive studies towards functional characterization of the gene, established that it is composed of 744 nucleotides and encodes a peptide of 247 amino acid residues. It possessed significant homology with AoGV and ClanGV with 87% identity at amino acid level. Multiple alignment data suggests that the C-terminus region of the gene had three different conserved regions. Time-course studies conducted in PiraGV-K infected P. rapae larvae revealed a significant upsurge of the transcript (134-fold) at 4 days post infection followed by a significant decline at the most advanced stages of infection. Anti-PiraGV-K granulin antibody was produced and western blot conducted with the infected larvae further confirmed the induction pattern with a protein of 30 kDa. Immunofluorescent staining showed a granulin-specific signal in fat body and integument of the infected larvae. Granulin-specific signals were noticed 2 days post infection with the eventual systemic spread of infection to the associated tracheal matrix witnessed at 4 days post infection. Immunogold labeling and electron microscopic studies further proved the cytopathological effects as the presence of numerous membrane-bound vesicles with nucleocapsids and abruption of intercellular junctions in fat body and hypertrophied cells in the integument.