RESUMEN

Serine protease cascades regulate important insect immune responses, including melanization and Toll pathway activation. In the context of melanization, central components of these cascades are clip domain serine proteases (CLIPs) including the catalytic, clip domain serine proteases (cSPs) and their non-catalytic homologs (cSPHs). Here, we define partially the structural hierarchy of An. gambiae cSPs of the CLIPB family, central players in melanization, and characterize their relative contributions to bacterial melanization and to mosquito susceptibility to bacterial infections. Using in vivo genetic analysis we show that the protease cascade branches downstream of the cSPs CLIPB4 and CLIPB17 into two branches one converging on CLIPB10 and the second on CLIPB8. We also show that the contribution of key cSPHs to melanization in vivo in response to diverse microbial challenges is more significant than any of the individual cSPs, possibly due to partial functional redundancy among the latter. Interestingly, we show that the key cSPH CLIPA8 which is essential for the efficient activation cleavage of CLIPBs in vivo is efficiently cleaved itself by several CLIPBs in vitro, suggesting that cSPs and cSPHs regulate signal amplification and propagation in melanization cascades by providing positive reinforcement upstream and downstream of each other.

Asunto(s)

Anopheles , Infecciones Bacterianas , Animales , Anopheles/genética , Anopheles/metabolismo , Anopheles/microbiología , Serina Proteasas , Serina Endopeptidasas/genética , Serina Endopeptidasas/química , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo

RESUMEN

Insect humoral immune responses are regulated in part by protease cascades, whose components circulate as zymogens in the hemolymph. In mosquitoes, these cascades consist of clip-domain serine proteases (cSPs) and/or their non-catalytic homologs, which form a complex network, whose molecular make-up is not fully understood. Using a systems biology approach, based on a co-expression network of gene family members that function in melanization and co-immunoprecipitation using the serine protease inhibitor (SRPN)2, a key negative regulator of the melanization response in mosquitoes, we identify the cSP CLIPB4 from the African malaria mosquito Anopheles gambiae as a central node in this protease network. CLIPB4 is tightly co-expressed with SRPN2 and forms protein complexes with SRPN2 in the hemolymph of immune-challenged female mosquitoes. Genetic and biochemical approaches validate our network analysis and show that CLIPB4 is required for melanization and antibacterial immunity, acting as a prophenoloxidase (proPO)-activating protease, which is inhibited by SRPN2. In addition, we provide novel insight into the structural organization of the cSP network in An. gambiae, by demonstrating that CLIPB4 is able to activate proCLIPB8, a cSP upstream of the proPO-activating protease CLIPB9. These data provide the first evidence that, in mosquitoes, cSPs provide branching points in immune protease networks and deliver positive reinforcement in proPO activation cascades.

Asunto(s)

Anopheles , Serpinas , Animales , Femenino , Inmunidad Humoral , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Serina Proteasas/genética , Serpinas/genética , Serpinas/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/metabolismo

RESUMEN

Serine protease cascades regulate important insect immune responses, including melanization and Toll pathway activation. In the context of melanization, central components of these cascades are clip domain serine proteases (CLIPs) including the catalytic, clip domain serine proteases (cSPs) and their non-catalytic homologs (cSPHs). Here, we define partially the structural hierarchy of An. gambiae cSPs of the CLIPB family, central players in melanization, and characterize their relative contributions to bacterial melanization and to mosquito susceptibility to bacterial infections. Using in vivo genetic analysis we show that the protease cascade branches downstream of the cSPs CLIPB4 and CLIPB17 into two branches one converging on CLIPB10 and the second on CLIPB8. We also show that the contribution of key cSPHs to melanization in vivo in response to diverse microbial challenges is more significant than any of the individual cSPs, possibly due to partial functional redundancy among the latter. Interestingly, we show that the key cSPH CLIPA8 which is essential for the efficient activation cleavage of CLIPBs in vivo is efficiently cleaved itself by several CLIPBs in vitro, suggesting that cSPs and cSPHs regulate signal amplification and propagation in melanization cascades by providing positive reinforcement upstream and downstream of each other.

RESUMEN

Clip-domain serine proteases (CLIPs) play important roles in insect innate immunity and development. Our previous studies indicated that CLIP13, an epidermis-specific gene, was involved in cuticle remodeling during molting and metamorphosis in the silkworm, Bombyx mori. However, the transcriptional regulatory mechanism and regulatory pathways of CLIP13 remained unclear. In the present study, we investigated CLIP13 expression and the regulation pathway controlled by 20-hydroxyecdysone (20E) in the silkworm. At the transcriptional level, expression of CLIP13 exhibited pronounced spatial and temporal specificity in different regions of the epidermis; homeodomain transcription factors POU-M2, antennapedia (Antp), and abdominal-B (Abd-B) showed similar expression change trends as CLIP13 in the head capsule, thorax, and abdomen, respectively. Furthermore, results of cell transfection assays, electrophoretic mobility shift assays, and chromatin immunoprecipitation demonstrated that POU-M2, Antp, and Abd-B were involved in the transcriptional regulation of CLIP13 by directly binding to their cis-response elements in CLIP13 promoter. RNA interference-mediated silencing of POU-M2, Antp, and Abd-B led to a decrease of CLIP13 expression in the head capsule, the epidermis of the 1st to 3rd thoracic segments and the 7th to 10th abdominal segments, respectively. Consistent with CLIP13, 20E treatment significantly upregulated expression of POU-M2, Antp, and Abd-B in the silkworm epidermis. Taken together, these data suggest that 20E positively regulates transcription of CLIP13 via homeodomain proteins POU-M2, Antp, and Abd-B in different regions of the silkworm epidermis during metamorphosis, thus affecting the molting process. Our findings provide new insight into the functions of homeodomain transcription factors in insect molting.

Asunto(s)

Bombyx , Abdomen , Animales , Bombyx/genética , Ecdisterona , Proteínas de Homeodominio/genética , Proteínas de Insectos/genética , Serina , Serina Proteasas/genética

RESUMEN

Clip domain serine protease (cSPs) play an important role in the innate immune defense of crustaceans. In this study, a clip domain serine protease (MncSP) and its alternative transcript (MncSP-isoform) were identified from Macrobrachium nipponense. The full-length cDNA sequences of MncSP and MncSP-isoform were 2447 and 2351 bp with open reading frames comprising 1497 and 1401 bp nucleotides and encoding 498 and 466 amino acids, respectively. The genome of MncSP had 10 exons and 9 introns. MncSP contained all 10 exons, whereas MncSP-isoform lacked the second exon. MncSP and MncSP-isoform contained a signal peptide, a clip domain, and a Tryp_SPc domain. Phylogenetic tree analysis showed that MncSP and MncSP-isoform clustered with cSPs from Palaemonidae. MncSP and MncSP-isoform were widely distributed in hemocytes, heart, hepatopancreas, gills, stomach, and intestine. The expression profiles of MncSP and MncSP-isoform in the hemocytes of M. nipponense changed after simulation by Vibrio parahaemolyticus or Staphylococcus aureus. The RNAi of MncSP could inhibit the expression of antimicrobial peptides (AMPs), including crustins and anti-lipopolysaccharide factors. Phenoloxidase activity was also down-regulated in MncSP-silenced prawns. This study indicated that MncSP participated in the synthesis of AMPs and the activation of prophenoloxidase.

Asunto(s)

Palaemonidae , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/metabolismo , Secuencia de Bases , Regulación de la Expresión Génica , Inmunidad Innata/genética , Filogenia , Isoformas de Proteínas/genética , Serina Proteasas/genética , Serina Proteasas/metabolismo , Instrumentos Quirúrgicos

RESUMEN

Clip-domain serine proteases (CLIPs), characterized by regulatory module clip domains, constitute an important serine protease family identified in insects and other arthropods. They participate in host immune response and embryonic development in a cascade-activated manner. Here, we present a genome-wide identification and expression analysis of CLIP genes in the silkworm, Bombyx mori. A total of 26 CLIP genes were identified in the silkworm genome. Bioinformatics analysis indicated that these CLIPs clustered into four subfamilies (CLIPA-D), and exhibit a close evolutionary relationship with CLIPs of Manduca sexta. Tissue expression profiling revealed that silkworm CLIP genes are mainly expressed in the integument, head, fat body, and hemocytes. Temporal expression profiles showed that 15 CLIP genes were predominantly expressed during the fifth-instar larval stage, early and later period of the pupal stage, and adult stage, whereas 10 CLIP genes were mainly expressed in the wandering stage and middle to later period of the pupal stage in the integument. Pathogens and 20-hydroxyecdysone (20E) induction analysis indicated that 14 CLIP genes were positively regulated by 20E, 9 were negatively regulated by 20E but positively regulated by pathogens, and 5 were positively regulated by both factors in the integument. Together, these results suggested that silkworm CLIP genes may play multiple functions in integument development, including melanization of new cuticle, molting and immune defense. Our data provide a comprehensive understanding of CLIP genes in the silkworm integument and lays a foundation for further functional studies of CLIP genes in the silkworm.

Asunto(s)

Proteínas de Artrópodos/genética , Bombyx/fisiología , Infecciones por Escherichia coli/inmunología , Escherichia coli/fisiología , Infecciones por Bacterias Grampositivas/inmunología , Micrococcus luteus/fisiología , Dominios Proteicos/genética , Serina Proteasas/genética , Animales , Proteínas de Artrópodos/metabolismo , Células Cultivadas , Ecdisterona/metabolismo , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Inmunidad/genética , Especificidad de Órganos , Serina Proteasas/metabolismo

RESUMEN

Clip domain serine proteases (CDSPs) participate in the extracellular signaling cascades of various biological processes such as innate immune responses in invertebrates. CDSP genes have been isolated from numerous invertebrates. Nevertheless, the enzymatic properties of mollusk CDSPs are poorly understood. In the present study, we demonstrated that the amino acid sequences of the trypsin-like serine protease purified from the digestive fluid of the sea hare, Aplysia kurodai resemble those of the unidentified CDSP-type protein (TPS3) of Aplysia californica predicted by genome analysis. The purified enzyme produced single 34 and 26.5 kDa bands on SDS-PAGE under non-reducing and reducing conditions, respectively. The 34-kDa band generated two amino-terminal sequences that were similar to the deduced sequences of the clip and catalytic domains of TPS3. The single amino-terminal sequence of the 26.5 kDa band showed a single sequence homologous to the catalytic domain. Thus, the purified enzyme consists of clip and catalytic domains bridged by disulfide linkage(s). The subsite specificity and inhibitor sensitivity of the purified enzyme were clearly distinct from those of horseshoe crab and silkworm CDSPs. A good substrate for the sea hare enzyme was pyroglutamyl-Arg-Thr-Lys-Arg-4-methyl-7-coumarylamide. The enzyme activity was strongly inhibited by aprotinin but not leupeptin. The physiological function of the enzyme in the digestive fluid remains to be determined.

Asunto(s)

Aplysia/enzimología , Sistema Digestivo/enzimología , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Animales , Aplysia/genética , Dominio Catalítico , Electroforesis en Gel de Poliacrilamida , Serina Endopeptidasas/genética , Especificidad por Sustrato

RESUMEN

Clip domain serine proteases (cSPs), a family of multifunctional proteins, play a crucial role in innate immune system. Here, we report the functional characterization of two clip domain serine proteases (PtcSP1 and PtcSP3) from the swimming crab Portunus trituberculatus. The recombinant N-terminal clip domains and the C-terminal SP-like domains of PtcSP1 and PtcSP3 were expressed in Escherichia coli system, and assayed for various biological functions: protease activity, antimicrobial activity, bacterial clearance and microbial-binding activity. The recombinant SP-like domains of PtcSP1 and PtcSP3 exhibited trypsin-like protease activity, while their recombinant clip domains showed strong antibacterial activity and could bind to bacteria and yeast, suggesting the potential roles of PtcSP1 and PtcSP3 in immune defense and pattern recognition. Unlike PtcSP3, PtcSP1 revealed the opsonic activity as shown by a higher bacterial clearance rate of Vibrio alginolyticus coated with the combination of the recombinant clip domain and SP-like domain of PtcSP1 as compared with V. alginolyticus only. Knockdown of PtcSP1 or PtcSP3 by RNA interference resulted in a significant decrease of total phenoloxidase (PO) activity in crab, suggesting that PtcSP1 and PtcSP3 are involved in the proPO system. In addition, suppression of PtcSP1 or PtcSP3 changed the expression of PtALFs and complement-like components. All these findings suggest that PtcSP1 and PtcSP3 are multifunctional immune molecules and perform different protective functions in crab defense.

Asunto(s)

Braquiuros/genética , Braquiuros/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Serina Proteasas/genética , Serina Proteasas/inmunología , Secuencia de Aminoácidos , Animales , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Perfilación de la Expresión Génica , Filogenia , Alineación de Secuencia , Serina Proteasas/química , Vibrio alginolyticus

RESUMEN

Clip-domain serine proteases (CLIPs) play crucial roles in insect development and innate immunity. In this study, we identified a CLIP gene (designated LsCLIP3) from the cigarette beetle Lasioderma serricorne. LsCLIP3 contains a 1,773-bp open reading frame (ORF) encoding a 390-amino-acid protein and shows a conserved clip domain and a trypsin-like serine protease domain. Phylogenetic analysis indicated that LsCLIP3 was orthologous to the CLIP-B subfamily. LsCLIP3 was prominently expressed in larva, pupa, and early adult stages. In larval tissues, it was highly expressed in the integument and fat body. The expression of LsCLIP3 was induced by 20-hydroxyecdysone. A similar induction was also found by peptidoglycans from Escherichia coli and Staphylococcus aureus. RNA interference (RNAi)-mediated silencing of LsCLIP3 disrupted larval-pupal molting and specifically reduced the expression of genes in 20-hydroxyecdysone synthesis and signaling pathway. The chitin amounts of LsCLIP3 RNAi larvae were greatly decreased, and expressions of six chitin metabolic-related genes were significantly reduced. Knockdown of LsCLIP3 increased larval sensitivity to Gram-negative and Gram-positive bacteria. There was significantly decreased expression of four antimicrobial peptide (AMP) genes. The results suggest that LsCLIP3 is an important component of the larva to pupa molt and for the immunity of L. serricorne.

RESUMEN

Clip-domain serine proteases (Clip-SPs) mediate innate immunity and embryonic development in insects. However, the function of Clip-SPs in Apis cerana cerana is little known. Here, a Clip-SP gene, AccSp1, was identified. AccSp1 was mainly detected in third and sixth day instar larvae, dark-eyed pupae, and adults (1and 30 days post-emergence). In addition, AccSp1 was expressed at its highest level in the venom gland and epidermis than tentacle, abdomen, muscle, honey sac, head, leg, chest, hemolymph, rectum, and midgut. AccSp1 was induced by 4, 24, and 44 °C; H2O2; CdCl2; HgCl2; and pesticides (paraquat, pyridaben, and methomyl) and was inhibited by UV light and cyhalothrin treatments. When adults that had been pretreated with dsRNA 6 h prior (knocking AccSp1 down) were challenged with Bacillus bombysepticus for 18 h, the survival rate of bees greatly decreased, the activity of PO (phenoloxidase) was reduced, revealing that AccSp1 may play a critical role in assisting bees to survive the microbial infection and participate in regulating PO activity. The antioxidant enzymatic activities of catalase, peroxidase, and superoxide dismutase; the contents of hydrogen peroxide and malondialdehyde; and the ratio of NADP+/NADPH were all lower in samples containing dsRNA-AccSp1 interference than in control groups, but the content of carbonyl was not significantly different. These findings suggest the knockdown of AccSp1 may influence melanization so that the antioxidant enzyme activities and the harmful metabolites decreased. These results collectively suggest that AccSp1 plays critical roles in abiotic stresses responses and resistance to pathogens.

Asunto(s)

Abejas/genética , Abejas/inmunología , Inmunidad Innata/genética , Proteínas de Insectos/genética , Serina Proteasas/genética , Estrés Fisiológico/genética , Secuencia de Aminoácidos , Animales , Abejas/enzimología , Abejas/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Metaboloma , Monofenol Monooxigenasa/metabolismo , Filogenia , Interferencia de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Serina Proteasas/química , Serina Proteasas/metabolismo , Análisis de Supervivencia

RESUMEN

Vibrio parahemolyticus (V. parahemolyticus) is a major pathogen for abalone, an important economical shellfish in coastal area of China. There is little known about the abalone innate immune system against pathogen infection. Clip-domain serine proteases (cSPs) are increasingly recognized to play important roles in host immune defense in invertebrates. In this study, we cloned a cSP (Hdh-cSP) from abalone (Haliotis discus hannai). We found out that Hdh-cSP was widely expressed in multiple tissues of abalone, with highest level in the immune-like organ, hepatopancreas. V. parahemolyticus infection induced significantly elevated expression of Hdh-cSP in addition to better-characterized innate immune component genes including Rel/NF-κB, allograft inflammatory factor (ALInFa), macrophage expressed protein (MEP) and caspase-8. Importantly, the silencing of Hdh-cSP reduced the expression of these genes, suggesting that Hdh-cSP was an upstream regulatory factor in V. parahemolyticus infection. Further analysis showed that apoptosis of hemocytes was inhibited when the transcription of Hdh-cSP was knocked down, suggesting that Hdh-cSP participated in cell apoptosis by regulation of caspase 8 expression in V. parahemolyticus infection. Therefore, our study established an important role of cSP in the innate immunity against V. parahemolyticus infection in abalone.

Asunto(s)

Gastrópodos/genética , Gastrópodos/inmunología , Regulación de la Expresión Génica/inmunología , Inmunidad Innata/genética , Serina Proteasas/genética , Serina Proteasas/inmunología , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Gastrópodos/química , Gastrópodos/enzimología , Perfilación de la Expresión Génica , Filogenia , Alineación de Secuencia , Serina Proteasas/química , Vibrio

RESUMEN

Clip domain serine protease homologs (SPHs) are positive and negative regulators of Anopheles gambiae immune responses mediated by the complement-like protein TEP1 against Plasmodium malaria parasites and other microbial infections. We have previously reported that the SPH CLIPA2 is a negative regulator of the TEP1-mediated response by showing that CLIPA2 knockdown (kd) enhances mosquito resistance to infections with fungi, bacteria, and Plasmodium parasites. Here, we identify another SPH, CLIPA14, as a novel regulator of mosquito immunity. We found that CLIPA14 is a hemolymph protein that is rapidly cleaved following a systemic infection. CLIPA14 kd mosquitoes elicited a potent melanization response against Plasmodium berghei ookinetes and exhibited significantly increased resistance to Plasmodium infections as well as to systemic and oral bacterial infections. The activity of the enzyme phenoloxidase, which initiates melanin biosynthesis, dramatically increased in the hemolymph of CLIPA14 kd mosquitoes in response to systemic bacterial infections. Ookinete melanization and hemolymph phenoloxidase activity were further increased after cosilencing CLIPA14 and CLIPA2, suggesting that these two SPHs act in concert to control the melanization response. Interestingly, CLIPA14 RNAi phenotypes and its infection-induced cleavage were abolished in a TEP1 loss-of-function background. Our results suggest that a complex network of SPHs functions downstream of TEP1 to regulate the melanization reaction.

Asunto(s)

Anopheles/metabolismo , Hemolinfa/metabolismo , Inmunidad Innata , Proteínas de Insectos/metabolismo , Serina Endopeptidasas/metabolismo , Animales , Animales Modificados Genéticamente , Anopheles/inmunología , Anopheles/microbiología , Anopheles/parasitología , Activación Enzimática , Escherichia coli/crecimiento & desarrollo , Escherichia coli/inmunología , Escherichia coli/aislamiento & purificación , Femenino , Técnicas de Silenciamiento del Gen/veterinaria , Hemolinfa/inmunología , Hemolinfa/microbiología , Proteínas de Insectos/antagonistas & inhibidores , Proteínas de Insectos/genética , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Melaninas/genética , Melaninas/metabolismo , Monofenol Monooxigenasa/genética , Monofenol Monooxigenasa/metabolismo , Plasmodium berghei/crecimiento & desarrollo , Plasmodium berghei/inmunología , Plasmodium berghei/aislamiento & purificación , Proteolisis , Interferencia de ARN , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , Serratia marcescens/crecimiento & desarrollo , Serratia marcescens/inmunología , Serratia marcescens/aislamiento & purificación , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/inmunología , Staphylococcus aureus/aislamiento & purificación , Análisis de Supervivencia , Regulación hacia Arriba

RESUMEN

Clip domain serine proteases (CLIPs), characterized by one or more conserved clip domains, are essential components of extracellular signalling cascades in various biological processes, especially in innate immunity and the embryonic development of insects. Additionally, CLIPs may have additional non-immune functions in insect development. In the present study, the clip domain serine protease gene Bombyx mori serine protease 95 (BmSP95), which encodes a 527-residue protein, was cloned from the integument of B. mori. Bioinformatics analysis indicated that BmSP95 is a typical CLIP of the subfamily D and possesses a clip domain at the N terminus, a trypsin-like serine protease (tryp_spc) domain at the C terminus and a conserved proline-rich motif between these two domains. At the transcriptional level, BmSP95 is expressed in the integument during moulting and metamorphosis, and the expression pattern is consistent with the fluctuating 20-hydroxyecdysone (20E) titre in B. mori. At the translational level, BmSP95 protein is synthesized in the epidermal cells, secreted as a zymogen and activated in the moulting fluid. Immunofluorescence revealed that BmSP95 is distributed into the old endocuticle in the moulting stage. The expression of BmSP95 was upregulated by 20E. Moreover, expression of BmSP95 was downregulated by pathogen infection. RNA interference-mediated silencing of BmSP95 led to delayed moulting from pupa to moth. These results suggest that BmSP95 is involved in integument remodelling during moulting and metamorphosis.

Asunto(s)

Bombyx/fisiología , Proteínas de Insectos/metabolismo , Muda , Serina Proteasas/metabolismo , Secuencia de Aminoácidos , Animales , Bacillus , Beauveria , Ecdisterona/metabolismo , Proteínas de Insectos/genética , Proteínas de Insectos/aislamiento & purificación , Larva/enzimología , Datos de Secuencia Molecular , Filogenia , Pupa/enzimología , Interferencia de ARN , Análisis de Secuencia de ADN , Serina Proteasas/genética , Serina Proteasas/aislamiento & purificación , Serratia marcescens

RESUMEN

Clip domain serine proteases (clip-SPs) play critical roles in various immune responses in arthropods, such as hemolymph coagulation, antimicrobial peptide (AMP) synthesis, cell adhesion and melanization. In the present study, we report the molecular and functional characterization of a clip domain serine protease (PtcSP2) from the swimming crab Portunus trituberculatus. The N-terminal clip domain and the C-terminal SP-like domain of PtcSP2 were expressed in Escherichia coli system, and assayed for their activities. Sequence similarity and phylogenetic analysis revealed that PtcSP2 may belong to the chymotrypsin family, which was confirmed by protease activity assay of the recombinant SP-like domain. The clip domain of PtcSP2 exhibited strong antibacterial activity and microbial-binding activity, suggesting the potential role in immune defense and recognition. Knockdown of PtcSP2 by RNA interference could significantly reduce PtcSP2 transcript levels, but neither decrease the total phenoloxidase (PO) activity in crab nor significantly alter the expression levels of serine protease inhibitors PtPLC and PtSerpin. These results indicate that PtcSP2 is not involved in the proPO system. However, suppression of PtcSP2 led to a significant change in the expression of AMP genes PtALFs and PtCrustin but not PtALF5. All these findings suggest that PtcSP2 is a multifunctional chymotrypsin-like serine protease and may participate in crab innate immunity by its antibacterial activity, immune recognition or regulation of AMP expression.

Asunto(s)

Braquiuros/enzimología , Quimasas/inmunología , Inmunidad Innata/genética , Secuencia de Aminoácidos , Animales , Péptidos Catiónicos Antimicrobianos/inmunología , Proteínas de Artrópodos/química , Proteínas de Artrópodos/genética , Proteínas de Artrópodos/inmunología , Braquiuros/clasificación , Braquiuros/genética , Braquiuros/inmunología , Catecol Oxidasa/genética , Catecol Oxidasa/inmunología , Quimasas/química , Quimasas/genética , Precursores Enzimáticos/genética , Precursores Enzimáticos/inmunología , Bacterias Gramnegativas/crecimiento & desarrollo , Bacterias Gramnegativas/inmunología , Bacterias Grampositivas/crecimiento & desarrollo , Bacterias Grampositivas/inmunología , Filogenia , Pichia/crecimiento & desarrollo , Pichia/inmunología , Alineación de Secuencia

RESUMEN

Exposure to entomopathogenic fungi is one approach for insect pest control. Little is known about the immune interactions between fungus and its insect host. Melanization is a prominent immune response in insects in defending against pathogens such as bacteria and fungi. Clip domain serine proteases in insect plasma have been implicated in the activation of prophenoloxidase, a key enzyme in the melanization. The relationship between host melanization and the infection by a fungus needs to be established. We report here that the injection of entomopathogenic fungus Beauveria bassiana induced both melanin synthesis and phenoloxidase activity in its host insect, the Asian corn borer, Ostrinia furnacalis (Guenée). qRT-PCR analysis showed several distinct patterns of expression of 13 clip-domain serine proteases in response to the challenge of fungi, with seven increased, two decreased, and four unchanged. Of special interest among these clip-domain serine protease genes are SP1 and SP13, the orthologs of Manduca sexta HP6 and PAP1 which are involved in the prophenoloxidase activation pathway. Recombinant O. furnacalis SP1 was found to activate proSP13 and induce the phenoloxidase activity in corn borer plasma. Additionally, SP13 was determined to directly cleave prophenoloxidase and therefore act as the prophenoloxidase activating protease. Our work thus reveals a biochemical mechanism in the melanization in corn borer associated with the challenge by B. bassiana injection. These insights could provide valuable information for better understanding the immune responses of Asian corn borer against B. bassiana.

Asunto(s)

Beauveria , Interacciones Huésped-Parásitos/fisiología , Proteínas de Insectos/metabolismo , Lepidópteros/enzimología , Lepidópteros/parasitología , Serina Proteasas/metabolismo , Animales , Beauveria/inmunología , Immunoblotting , Lepidópteros/inmunología , Melaninas/metabolismo , Proteínas Asociadas a Pancreatitis , Control Biológico de Vectores/métodos , Reacción en Cadena de la Polimerasa

RESUMEN

The phenoloxidase (PO) activation system plays an important role in insect innate immunity, particularly in wound healing and pathogen defense. A key member of this system is prophenoloxidase-activating protease (PAP), which is the direct activator of prophenoloxidase (proPO). Despite their importance in the insect PO activation system, content of studies is limited. In this article, we identify two complementary DNAs (cDNAs), PxPAPa and PxPAPb, encoding possible PAPs, from immunized larval hemocytes of the diamondback moth, Plutella xylostella (L.), by RACE method. PxPAPa is 1,149-bp long and encodes a 382-residue open reading frame (ORF) with a predicted 17-residue signal peptide, a clip domain, and a Tryp_Spc domain. PxPAPb is 1,650-bp long and encodes a 440-residue ORF with a predicted 20-residue signal peptide, two clip domains, and a Tryp_Spc domain. PxPAPa and PxPAPb have a high sequence similarity to Manduca sexta (L.) PAP1 and PAP3, respectively. We also examined the transcript patterns of PxPAPa, PxPAPb, and pxPAP3, another clip-domain serine protease gene, response to different microbial challenges by using real-time quantitative polymerase chain reaction. The results show that the transcript abundance of PxPAPa is significantly increased by Micrococcus luteus and Escherichia coli but not Candida albicans. PxPAPb is induced only by Mi. luteus, whereas pxPAP3 could be induced by all the microbes in the test, but the transcript patterns of Mi. luteus, E. coli, and C. albicans are completely different. This study provides new insights into the molecular events that occur during the immune response, particularly melanization cascade that is involved in encapsulation and nodulation of pathogen or parasite invaders via hemocytes in host insects.

Asunto(s)

Inmunidad Innata/genética , Proteínas de Insectos/metabolismo , Mariposas Nocturnas/fisiología , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Hemocitos/enzimología , Interacciones Huésped-Patógeno/inmunología , Proteínas de Insectos/genética , Larva/enzimología , Datos de Secuencia Molecular , Proteínas Asociadas a Pancreatitis , Filogenia , Serina Endopeptidasas/genética