An inhibitor of NF-kB encoded in Cotesia plutella bracovirus inhibits expression of antimicrobial peptides and enhances pathogenicity of Bacillus thuringiensis

Sony Shrestha,Hosanna H. Kim,김용균 한국응용곤충학회 2009 Journal of Asia-Pacific Entomology Vol.12 No.4

Upon parasitization by an endoparasitoid wasp, Cotesia plutellae, the diamondback moth, Plutella xylostella, exhibits significant immunosuppression. A bracovirus (CpBV) symbiotic C. plutellae, has been regarded as a main parasitic factor due to acute and persistent expression of various encoded genes. Inhibitor-kB genes (CpBV-IkB) are homologous to cactus gene of Drosophila and are found in CpBV genome. However, their function in parasitism was unknown. Here, we tested hypothesis that CpBV-IkB may interrupt nuclear factor kappa B (NF-kB) to inhibit its translocation into the nucleus, resulting in the suppression of antimicrobial peptide synthesis. A CpBV-IkB was cloned into an expression vector and micro-injected into nonparasitized larvae. The transiently expressed CpBV-IkB in P. xylostella inhibited the expression of hemolin, but did not inhibit the expression of lysozyme or cecropin. This inhibitory activity of CpBV-IkB was more evident in a non-natural host, Spodoptera exigua, where both lysozyme and cecropin were inhibited. A recombinant Autographa californica multiple nucleopolyhedrosis virus (AcMNPV) was constructed by recombining CpBV-IkB gene under an early expression promoter. The budded form of the recombinant virus was injected into the hemocoel, while polyhedral form of the recombinant virus was orally administered to the P. xylostella larvae. In both treatments, the expression of CpBV-IkB encoded in the baculovirus was confirmed by reverse transcriptase (RT)-PCR. When the recombinant virus was mixed with Bacillus thuringiensis subsp. kurstaki (Bt), the bacterial pathogenicity was significantly enhanced in a dose-dependent manner, compared to a Bt mixture with an AcMNPV recombined with an enhanced green fluorescence protein gene. These results suggest that the inhibitory effect of CpBV-IkB on the production of antimicrobial peptide results in the enhanced pathogenicity of Bt. Upon parasitization by an endoparasitoid wasp, Cotesia plutellae, the diamondback moth, Plutella xylostella, exhibits significant immunosuppression. A bracovirus (CpBV) symbiotic C. plutellae, has been regarded as a main parasitic factor due to acute and persistent expression of various encoded genes. Inhibitor-kB genes (CpBV-IkB) are homologous to cactus gene of Drosophila and are found in CpBV genome. However, their function in parasitism was unknown. Here, we tested hypothesis that CpBV-IkB may interrupt nuclear factor kappa B (NF-kB) to inhibit its translocation into the nucleus, resulting in the suppression of antimicrobial peptide synthesis. A CpBV-IkB was cloned into an expression vector and micro-injected into nonparasitized larvae. The transiently expressed CpBV-IkB in P. xylostella inhibited the expression of hemolin, but did not inhibit the expression of lysozyme or cecropin. This inhibitory activity of CpBV-IkB was more evident in a non-natural host, Spodoptera exigua, where both lysozyme and cecropin were inhibited. A recombinant Autographa californica multiple nucleopolyhedrosis virus (AcMNPV) was constructed by recombining CpBV-IkB gene under an early expression promoter. The budded form of the recombinant virus was injected into the hemocoel, while polyhedral form of the recombinant virus was orally administered to the P. xylostella larvae. In both treatments, the expression of CpBV-IkB encoded in the baculovirus was confirmed by reverse transcriptase (RT)-PCR. When the recombinant virus was mixed with Bacillus thuringiensis subsp. kurstaki (Bt), the bacterial pathogenicity was significantly enhanced in a dose-dependent manner, compared to a Bt mixture with an AcMNPV recombined with an enhanced green fluorescence protein gene. These results suggest that the inhibitory effect of CpBV-IkB on the production of antimicrobial peptide results in the enhanced pathogenicity of Bt.

Factors Affecting the Activation of Hemolymph Prophenoloxidaseof Spodoptera exigua (Lepidoptera: Noctuidae)

Sony Shrestha,김용균 한국응용곤충학회 2007 Journal of Asia-Pacific Entomology Vol.10 No.2

In insect immunity, phenoloxidase (PO) plays an important role in the processes during melanotic encapsulation and phagocytosis. However, uncontrolled PO activity is known to be fatal due to its toxic catalytic products. Thus, the activation of prophenoloxidase (proPO) to PO must be controlled. This study showed that at least three factors were involved in the proPO activation pathway of Spodoptera exigua. Most hemolymph PO activity of S. exigua was detected in hemocytes. The de novo synthesis of proPO was a factor by which PO activity levels were determined in hemolymph, as PO activity was inhibited in a dose-dependent manner by cycloheximide, a eukaryotic protein translation inhibitor. The second factor was derived from serine protease (s) becasue only serine protease inhibitors prevented proPO activation, while other proteases did not. In addition, eicosanoids were implicated in proPO activation, because dexamethasone, an inhibitor of phospholipase A2, inhibited proPO activation. These results indicate that the activation of proPO in S. exigua is controlled at both transcriptional and posttranscriptional levels.

Differential pathogenicity of two entomopathogenic bacteria, Photorhabdus temperata subsp. temperata and Xenorhabdus nematophila against the red flour beetle, Tribolium castaneum

Sony Shrestha,김용균 한국응용곤충학회 2010 Journal of Asia-Pacific Entomology Vol.13 No.3

Two entomopathogenic bacteria, Photorhabdus temperata subsp. temperata (Ptt) and Xenorhabdus nematophila (Xn), are symbiotically associated with the nematodes, Heterorhabdis megidis and Steinernema carpocapsae,respectively. There is little information on natural host ranges of the nematodes, but a significant difference in pathogenicity was observed between these two bacteria against the red flour beetle, Tribolium castaneum, in which Ptt exhibited more than six times higher pathogenicity than Xn. The pathogenic difference was not due to their inhibitory effect on phospholipase A2 activity that is required for expression of immune response of T. castaneum. The culture broths of both bacterial species had insecticidal activities when injected into the hemocoel. When the bacterial culture broths were fractionated into aqueous and organic extracts, most insecticidal activity remained in the aqueous extracts. The aqueous extracts of two bacteria contained proteins which showed different profiles.

Two chemical derivatives of bacterial metabolites suppress cellular immune responses and enhance pathogenicity of Bacillus thuringiensis against the diamondback moth, Plutella xylostella

Sony Shrestha,홍용표,Yonggyun Kim 한국응용곤충학회 2010 Journal of Asia-Pacific Entomology Vol.13 No.1

Eicosanoids mediate insect immune responses, especially against bacterial infection. Phospholipase A2(PLA2) catalyzes the committed step of the eicosanoid biosynthesis pathway. Three PLA2 inhibitors have been identified from metabolites of an entomopathogenic bacterium, Xenorhabdus nematophila:benzylideneacetone (BZA), Pro-Tyr (PY), and acetylated Phe-Gly-Val (Ac-FGV). Interestingly, they share benzenepropane as a core chemical structure. We analyzed the functional significance of the core structure using structural derivatives. Removing a phenyl ring from PY resulted in significant loss of the PLA2inhibitory activity, as seen in a Pro-Ala derivative. Though the p-hydroxyl group was not critical in PY as seen in Pro-Phe derivative, its addition to BZA resulted in significant loss of inhibitory activity. Some alterations of structures other than the core structure increased PLA2-inhibitory activity in some derivatives, including Ala-Tyr (AY) and Phe-Gly-Val (FGV) derivatives. Using these selected derivatives,we further analyzed synergistic effects on pathogenicity of Bacillus thuringiensis (Bt) against the second instar larvae of Plutella xylostella. These two derivatives significantly enhanced the Bt pathogenicity. This study introduces two novel compounds that inhibit PLA2 and suggests their application in combination with Bt to control P. xylostella.

An inhibitor of NF-kB encoded in Cotesia plutella bracovirus inhibits expression of antimicrobial peptides and enhances pathogenicity of Bacillus thuringiensis

Shrestha, Sony,Kim, Hosanna H.,Kim, Yong-Gyun 한국응용곤충학회 2009 Journal of Asia-Pacific Entomology Vol. No.

Upon parasitization by an endoparasitoid wasp, Cotesia plutellae, the diamondback moth, Plutella xylostella, exhibits significant immunosuppression. A bracovirus (CpBV) symbiotic C. plutellae, has been regarded as a main parasitic factor due to acute and persistent expression of various encoded genes. Inhibitor-kB genes (CpBV-IkB) are homologous to cactus gene of Drosophila and are found in CpBV genome. However, their function in parasitism was unknown. Here, we tested hypothesis that CpBV-IkB may interrupt nudear factor kappa B (NF-kB) to inhibit its translocation into the nudeus, resulting in the suppression of antimicrobial peptide synthesis. A CpBV-IkB was doned into an expression vector and micro-injected into nonparasitized larvae. The transiently expressed CpBV-IkB in P. xylostella inhibited the expression of hemolin, but did not inhibit the expression of lysozyme or cecropin. This inhibitory activity of CpBV-IkB was more evident in a non-natural host, Spodoptera exigua, where both lysozyme and cecropin were inhibited. A recombinant Autographa californica multiple nudeopolyhedrosis virus (AcMNPV) was constructed by recombining CpBV-IkB gene under an early expression promoter. The budded form of the recombinant virus was injected into the hemocoel, while polyhedral form of the recombinant virus was orally administered to the P. xylostella larvae. In both treatments, the expression of CpBV-IkB encoded in the baculovirus was confirmed by reverse transcriptase (RT)-PCR. When the recombinant virus was mixed with Bacillus thuringiensis subsp. kurstaki (Bt), the bacterial pathogenicity was significantly enhanced in a dose-dependent manner, compared to a Bt mixture with an AcMNPV recombined with an enhanced green fluorescence protein gene. These results suggest that the inhibitory effect of CpBV-IkB on the production of antimicrobial peptide results in the enhanced pathogenicity of Bt.

Cloning of phospholipase A2 gene and its role in mediating cellular immune response in red flour beetle, Tribolium castaneum

Sony Shrestha,Yoonseong Park,Yonggyun Kim 한국응용곤충학회 2008 한국응용곤충학회 학술대회논문집 Vol.2008 No.05

Phospholipase A2 (PLA2) is the committed catalytic step of eicosanoid biosynthesis, which has been a common molecular target of several entomopathogens to induce insect immunosuppression. Despite critical importance of PLA2 in insect immunity, its gene structure was not known. This study identified insect PLA2 gene associated with immune reactions in the red flour beetle, Tribolium castaneum. Based on a previous study that an immune-associated PLA2 in insect is secretory type of PLA2 (sPLA2), five highly matched cDNA sequences were obtained from T. castaneum genome database using an sPLA2 sequence probe encoded in Drosophila melanogaster. The expressions of these five putative PLA2 were confirmed by reverse transcriptase-polymerase chain reaction. Out of five genes, one PLA2 gene called TcPLA2B was chosen because it showed specific expression in hemocyte and fat body. TcPLA2B was cloned and expressed in Escherichia coli and its protein was purified. The purified TcPLA2B showed PLA2enzyme activity, which was specifically inhibited by bromophenacyl bromide (a specific sPLA2inhibitor) and dithiothreitol (reducing agent of disulfide bond). It was sensitive to pH (optimum at pH 6.0) and reaction temperature (optimum at 10-30°C), and calcium dependency. An immunofluorescence assay indicated that TcPLA2B was localized near to cellular membrane of the cytosol in the hemocytes of T. castaneum at immune chanlenge. Double-stranded RNA (dsRNA) of TcPLA2B-treated larvae showed knockdown of its mRNA expression and did not form hemocyte nodule formation, while control larvae could exhibit time- and bacterial dose-dependent nodule formation in response to bacterial challenge. Addition of arachidonic acid (the catalytic product of PLA2) to the dsRNA-treated larvae rescued the inhibition of nodule formation. These results suggest that TcPLA2B gene is associated with insect immune reaction.

Diagnostic molecular markers of six lepidopteran insect pests infesting apples in Korea

Shrestha Sony,Md. Abdul Alim,Sangwon Kim,Minsoo Kwon,Dongkyun Lee,김용균 한국응용곤충학회 2009 Journal of Asia-Pacific Entomology Vol.12 No.2

Two molecular identification techniques for differentiating six lepidopteran pests infesting apples in Korea are presented. These six species include two internal fruit feeders (Grapholita molesta and Carposina sasakii), two leaf rollers (Adoxophyes sp. and Archips breviplicanus) and two leaf miners (Phyllonorycter ringoniella and Lyonetia prunifoliella). All species occur until near harvest and reduce apple production. A 489 bp fragment of mitochondrial cytochrome oxidase subunit I (COI) was sequenced in these six species. The sequence was used to select species-specific restriction enzyme sites and to design diagnostic polymerase chain reaction (PCR) primers, resulting in the development of restriction fragment length polymorphism (RFLP)-PCR and diagnostic PCR. These methods were reliable and rapid in the identification of these six species. Two molecular identification techniques for differentiating six lepidopteran pests infesting apples in Korea are presented. These six species include two internal fruit feeders (Grapholita molesta and Carposina sasakii), two leaf rollers (Adoxophyes sp. and Archips breviplicanus) and two leaf miners (Phyllonorycter ringoniella and Lyonetia prunifoliella). All species occur until near harvest and reduce apple production. A 489 bp fragment of mitochondrial cytochrome oxidase subunit I (COI) was sequenced in these six species. The sequence was used to select species-specific restriction enzyme sites and to design diagnostic polymerase chain reaction (PCR) primers, resulting in the development of restriction fragment length polymorphism (RFLP)-PCR and diagnostic PCR. These methods were reliable and rapid in the identification of these six species.

Various Eicosanoids Modulate the Cellular and Humoral Immune Responses of the Beet Armyworm, <i>Spodoptera exigua</i>

SHRESTHA, Sony,KIM, Yonggyun Japan Society for Bioscience, Biotechnology, and A 2009 Bioscience, biotechnology, and biochemistry Vol.73 No.9

<P>Cyclooxygenase (COX) and lipoxygenase (LOX) can catalyze the oxidation of C20 fatty acids to produce certain eicosanoids, which play roles in mediating immune responses in insects. Despite their critical role in insect immunity, there have been few studies of the unique effects of different eicosanoids on immune responses. This study analyzed cellular and humoral immune responses of the beet armyworm, <I>Spodoptera exigua</I>, using seven eicosanoids selected from two major eicosanoid subgroups: prostaglandin (PG) and leukotriene (LT), derived from catalytic activities of COX and LOX respectively. Upon bacterial challenge, all seven eicosanoids (PGA<SUB>1</SUB>, PGB<SUB>2</SUB>, PGD<SUB>2</SUB>, PGE<SUB>1</SUB>, PGE<SUB>2</SUB>, PGF<SUB>1α</SUB>, and LTB<SUB>4</SUB>) significantly induced hemocyte nodulation and phagocytosis in the presence of dexamethasone, an eicosanoid biosynthesis inhibitor. However, only PGs induced cell lysis of oenocytoids to release prophenoloxidase, which resulted in an increase in phenoloxidase activity. These seven eicosanoids also induced expression of humoral immune-associated genes, including prophenoloxidase, serpin, dopa decarboxylase, cecropin, and lysozyme, in which PGB<SUB>2</SUB> and PGE<SUB>1</SUB> did not induce gene expression of prophenoloxidase. To understand the interactions between different eicosanoids, mixture effects of these eicosanoids were compared with their individual eicosanoid effects on mediating nodule formation in response to bacterial challenge. All six single PGs showed increases in nodule formation in a dose-dependent manner without significant difference among the different types. LTB<SUB>4</SUB> was more potent than the tested PGs in mediating the cellular immune response. At low doses, all combinations of two eicosanoids showed significant additive effects on nodule formation. These results indicate that immune target cells, such as hemocyte and fat body, of <I>S. exigua</I> can respond to different COX and LOX products to express cellular and humoral immune responses, and their overlapping, additive effects on nodulation suggest that in target cells, these eicosanoids share a hypothetical common eicosanoid signal pathway.</P>

Development of Novel Insecticidal Compounds Using Eicosanoid Biosynthesis Inhibitors Originated from Entomopathogenic Bacteria

Sony Shrestha,Yonggyun Kim 한국응용곤충학회 2009 한국응용곤충학회 학술대회논문집 Vol.2009 No.05

An entomopathogenic bacterium, Xenorhabdus nematophila, induces an immunosuppression by inhibiting phospholipase A2 (PLA2), which results in a fatal septicemia. PLA2 is an enzyme responsible for eicosanoid biosynthesis and the pathogenic molecular target of this bacterium. A PLA2 gene of the red flour beetle, Tribolium castaneum, was expressed in Escherichia coli. The recombinant T. castaneum PLA2 (TcPLA2) showed enzyme activity, which was specifically inhibited by bromophenacyl bromide (specific inhibitor to secretory PLA2) and ditheothreitol (reducing agent of disulfide bond). It was sensitive to pH (optimum at pH 7.0), temperature (optimum at 30°C), substrate specificity and calcium dependency. X. nematophila released compound(s) inhibiting TcPLA2during its stationary growth phase. The active compound (s) was resistant to heat treatment and could be extracted separately into both organic and aqueous phases. This PLA2 inhibitory fraction showed significant effect on immunosuppression of T. castaneum. These results suggest there may be several PLA2 inhibitors synthesized by X. nematophila and released into culture broth. The recombinant TcPLA2 was also used to screen potent PLA2 inhibitor compounds, which were designed based on a common chemical structure (a pentenebenzene ring) of two peptide inhibitors, proline-tyrosine (PY) and acetylated phenylalanine-glycine-valine (AcFGV). Alterations were made on amino acid sequence or specific functional groups on the pentenebenzene ring. Among 7 different peptides, AY and FGV showed the most potent effects on TcPLA2activity and also resulted in significant reductions in hemocyte spreading behavior of Plutella xylostella. The potent candidate molecules would be applied to control various insect pests to be developed into novel insecticides.

An Entomopathogenic Bacterium, Xenorhabdus nematophila, suppresses Hemocyte Phagocytosis of Spodoptera exigua by inhibiting Phospholipase A2

( Sony Shrestha ),( Yong Gyun Kim ) 한국응용곤충학회 2006 한국응용곤충학회 학술대회논문집 Vol.2006 No.9