RESUMO
Microbial production of hyaluronic acid (HA) is an area of research that has been gaining attention in recent years due to the increasing demand for this biopolymer for several industrial applications. Hyaluronic acid is a linear, non-sulfated glycosaminoglycan that is widely distributed in nature and is mainly composed of repeating units of N-acetylglucosamine and glucuronic acid. It has a wide and unique range of properties such as viscoelasticity, lubrication, and hydration, which makes it an attractive material for several industrial applications such as cosmetics, pharmaceuticals, and medical devices. This review presents and discusses the available fermentation strategies to produce hyaluronic acid.
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Acetilglucosamina , Ácido Hialurônico , Fermentação , Fenômenos Químicos , Ácido GlucurônicoRESUMO
We report the draft genome sequence of Agarivorans aestuarii strain ZMCS4, isolated from Cladostephus spongiosus. The assembled genome consists of 4.5 Mbp, comprising 25 contigs and 4,128 coding sequences. This genome will provide insights into further studies on relevant CAZymes involved in the hydrolysis of algal cell walls.
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The Japanese beetle Popillia japonica is a pest insect that feeds on hundreds of species of wild and cultivated plants including important fruit, vegetable, and field crops. Native to Japan, the pest has invaded large areas of the USA, Canada, the Azores (Portugal), Italy, and Ticino (Switzerland), and it is considered a priority for control in the European Union. We determined the complete mitochondrial genome sequence in 86 individuals covering the entire distribution of the species. Phylogenetic analysis supports a major division between South Japan and Central/North Japan, with invasive samples coming from the latter. The origin of invasive USA samples is incompatible, in terms of the timing of the event, with a single introduction, with multiple Japanese lineages having been introduced and one accounting for most of the population expansion locally. The origin of the two invasive European populations is compatible with two different invasions followed by minimal differentiation locally. Population analyses provide the possibility to estimate the rate of sequence change from the data and to date major invasion events. Demographic analysis identifies a population expansion followed by a period of contraction prior to the invasion. The present study adds a time and demographic dimension to available reconstructions.
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Besouros , Genoma Mitocondrial , Animais , Besouros/genética , Filogenia , Plantas/genética , DemografiaRESUMO
Popillia japonica (Coleoptera: Scarabaeidae), is an emerging invasive pest in Europe and America. In the Azores, this pest was first found on Terceira Island during the sixties and soon spread to other islands. The rate of infestation differs between islands, and we hypothesized that microbiome composition could play a role. Therefore, we sampled 3rd instar larvae and soil from sites with high and low infestation rates to analyze the microbiome using next-generation sequencing. We analyzed twenty-four 16S DNA libraries, which resulted in 3278 operational taxonomic units. The alpha and beta diversity of the soil microbiome was similar between sites. In contrast, the larvae from high-density sites presented a higher bacterial gut diversity than larvae from low-density sites, with biomarkers linked to plant digestion, nutrient acquisition, and detoxification. Consequently, larvae from high-density sites displayed several enriched molecular functions associated with the families Ruminococcaceae, Clostridiaceae and Rikenellaceae. These bacteria revealed a supportive function by producing several CAZyme families and other proteins. These findings suggest that the microbiome must be one drive for the increase in P. japonica populations, thus providing a checkpoint in the establishment and spread of this pest.
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Large-scale production of microalgae and their bioactive compounds has steadily increased in response to global demand for natural compounds. Spirulina, in particular, has been used due to its high nutritional value, especially its high protein content. Promising biological functions have been associated with Spirulina extracts, mainly related to its high value added blue pigment, phycocyanin. Phycocyanin is used in several industries such as food, cosmetics, and pharmaceuticals, which increases its market value. Due to the worldwide interest and the need to replace synthetic compounds with natural ones, efforts have been made to optimize large-scale production processes and maintain phycocyanin stability, which is a highly unstable protein. The aim of this review is to update the scientific knowledge on phycocyanin applications and to describe the reported production, extraction, and purification methods, including the main physical and chemical parameters that may affect the purity, recovery, and stability of phycocyanin. By implementing different techniques such as complete cell disruption, extraction at temperatures below 45 °C and a pH of 5.5-6.0, purification through ammonium sulfate, and filtration and chromatography, both the purity and stability of phycocyanin have been significantly improved. Moreover, the use of saccharides, crosslinkers, or natural polymers as preservatives has contributed to the increased market value of phycocyanin.
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Steinernema carpocapsae is an entomopathogenic nematode (EPN) that rapidly infects and kills a wide range of insect hosts and has been linked to host immunosuppression during the initial stages of infection. The lethal nature of S. carpocapsae infections has previously been credited to its symbiotic bacteria; however, it has become evident that the nematodes are able to effectively kill their hosts independently through their excretion/secretion products (ESPs). Here we examined how the adult Drosophila melanogaster immune system is modulated in response to S. carpocapsae ESPs in an attempt to ascertain individual pathogenic contributions of the isolated compound. We found that the S. carpocapsae ESPs decrease the survival of D. melanogaster adult flies, they induce the expression of certain antimicrobial peptide-encoding genes, and they cause significant reduction in phenoloxidase enzyme activity and delay in the melanization response in males flies. We also report that S. carpocapsae ESPs affect hemocyte numbers in both male and female individuals. Our results indicate the manipulative role of EPN ESPs and reveal sex-specific differences in the host response against nematode infection factors. These findings are beneficial as they promote our understanding of the molecular basis of nematode pathogenicity and the parasite components that influence nematode-host interactions.
Assuntos
Infecções por Nematoides , Rabditídios , Animais , Drosophila melanogaster/genética , Feminino , Interações Hospedeiro-Parasita , Imunidade , MasculinoRESUMO
Entomopathogenic nematodes are used as biological control agents against a broad range of insect pests. We ascribed the pathogenicity of these organisms to the excretory/secretory products (ESP) released by the infective nematode. Our group characterized different virulence factors produced by Steinernema carpocapsae that underlie its success as an insect pathogen. A novel ShK-like peptide (ScK1) from this nematode that presents high sequence similarity with the ShK peptide from a sea anemone was successfully produced recombinantly in Escherichia coli. The secondary structure of ScK1 appeared redox-sensitive, exhibiting a far-UV circular dichroism spectrum consistent with an alpha-helical secondary structure. Thermal denaturation of the ScK1 allowed estimating the melting temperature to 59.2 ± 0.1 °C. The results from toxicity assays using Drosophila melanogaster as a model show that injection of this peptide can kill insects in a dose-dependent manner with an LD50 of 16.9 µM per adult within 24 h. Oral administration of the fusion protein significantly reduced the locomotor activity of insects after 48 h (p < 0.05, Tukey's test). These data show that this nematode expresses insecticidal peptides with potential as next-generation insecticides.
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Drosophila melanogaster , Nematoides , Animais , Insetos , Agentes de Controle Biológico , VirulênciaRESUMO
Steinernema carpocapsae is an insect parasitic nematode used in biological control, which infects insects penetrating by mouth and anus and invading the hemocoelium through the midgut wall. Invasion has been described as a key factor in nematode virulence and suggested to be mediated by proteases. A serine protease cDNA from the parasitic stage was sequenced (sc-sp-1); the recombinant protein was produced in an Escherichia coli system, and a native protein was purified from the secreted products. Both proteins were confirmed by mass spectrometry to be encoded by the sc-sp-1 gene. Sc-SP-1 has a pI of 8.7, a molecular mass of 27.3 kDa, a catalytic efficiency of 22.2 × 10(4) s(-1) m(-1) against N-succinyl-Ala-Ala-Pro-Phe-pNA, and is inhibited by chymostatin (IC 0.07) and PMSF (IC 0.73). Sc-SP-1 belongs to the chymotrypsin family, based on sequence and biochemical analysis. Only the nematode parasitic stage expressed sc-sp-1. These nematodes in the midgut lumen, prepared to invade the insect hemocoelium, expressed higher levels than those already in the hemocoelium. Moreover, parasitic nematode sense insect peritrophic membrane and hemolymph more quickly than they do other tissues, which initiates sc-sp-1 expression. Ex vivo, Sc-SP-1 was able to bind to insect midgut epithelium and to cause cell detachment from basal lamina. In vitro, Sc-SP-1 formed holes in an artificial membrane model (Matrigel), whereas Sc-SP-1 treated with PMSF did not, very likely because it hydrolyzes matrix glycoproteins. These findings highlight the S. carpocapsae-invasive process that is a key step in the parasitism thus opening new perspectives for improving nematode virulence to use in biological control.
Assuntos
Proteínas de Helminto/química , Insetos/parasitologia , Nematoides/enzimologia , Serina Proteases/química , Sequência de Aminoácidos , Animais , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Hemolinfa/parasitologia , Dados de Sequência Molecular , Nematoides/genética , Nematoides/patogenicidade , Oligopeptídeos/química , Controle Biológico de Vetores/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Serina Proteases/genética , Serina Proteases/metabolismoRESUMO
Fibrinolytic enzymes with a direct mechanism of action and safer properties are currently requested for thrombolytic therapy. This paper reports on a new enzyme capable of degrading blood clots directly without impairing blood coagulation. This enzyme is also non-cytotoxic and constitutes an alternative to other thrombolytic enzymes known to cause undesired side effects. Twenty-four Bacillus isolates were screened for production of fibrinolytic enzymes using a fibrin agar plate. Based on produced activity, isolate S127e was selected and identified as B. subtilis using the 16S rDNA gene sequence. This strain is of biotechnological interest for producing high fibrinolytic yield and consequently has potential in the industrial field. The purified fibrinolytic enzyme has a molecular mass of 27.3 kDa, a predicted pI of 6.6, and a maximal affinity for Ala-Ala-Pro-Phe. This enzyme was almost completely inhibited by chymostatin with optimal activity at 48°C and pH 7. Specific subtilisin features were found in the gene sequence, indicating that this enzyme belongs to the BPN group of the S8 subtilisin family and was assigned as AprE127. This subtilisin increased thromboplastin time by 3.7% (37.6 to 39 s) and prothrombin time by 3.2% (12.6 to 13 s), both within normal ranges. In a whole blood euglobulin assay, this enzyme did not impair coagulation but reduced lysis time significantly. Moreover, in an in vitro assay, AprE127 completely dissolved a thrombus of about 1 cc within 50 min and, in vivo, reduced a thrombus prompted in a rat tail by 11.4% in 24 h compared to non-treated animals.
Assuntos
Bacillus subtilis/enzimologia , Proteínas de Bactérias/isolamento & purificação , Fibrinolíticos/isolamento & purificação , Subtilisinas/isolamento & purificação , Trombose/tratamento farmacológico , Animais , Bacillus subtilis/química , Bacillus subtilis/genética , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/química , Células Sanguíneas/efeitos dos fármacos , Células Sanguíneas/fisiologia , Coagulação Sanguínea/efeitos dos fármacos , Feminino , Fibrinolíticos/administração & dosagem , Fibrinolíticos/química , Hemólise/efeitos dos fármacos , Humanos , Concentração de Íons de Hidrogênio , Cinética , Peso Molecular , Tempo de Protrombina , Ratos , Ovinos , Subtilisinas/administração & dosagem , Subtilisinas/química , Trombose/fisiopatologiaRESUMO
BACKGROUND: In order to identify molecules necessary for the invasion of the mosquito midgut epithelium by plasmodia, interaction assays between both these structures were devised. MATERIALS AND METHODS: Enrichment of Plasmodium berghei ookinetes was first carried out using a double 5-17% gradient Nycodenz cushion, which resulted in an enrichment factor of over 800%. Viability of these ookinetes was confirmed by membrane feeding mosquito infection assays, showing no decrease in infection prevalence or intensity, and suggesting that putative surface proteins necessary for the invasion were unaffected. RESULTS: Protein interaction assays between mosquito midgut and ookinete extracts were optimized yielding Anopheles gambiae mosquito midgut proteins of >220, 200 and 48 kDa that could bind biotinylated P. berghei ookinete extracts, and P. berghei ookinete proteins of 48-45 kDa that could bind biotinylated An. gambiae midgut proteins. Using an An. gambiae midgut biotinylated extract linked to a streptavidin-agarose matrix, P. berghei ookinete proteins of approximately 116, 45 and 21 kDa were obtained. This protein chromotography pull-down assay was reproducibly repeated and spots from 2D eletrophoretic separation were analysed by mass spectrometry. For one spot, a significant match with a putative erythrocyte binding protein from P. falciparum (Pf EBA-165) was obtained. This protein belongs to the erythrocyte binding superfamily of the merozoite stage that is involved in the invasion process of the erythrocytes. CONCLUSION: Our findings suggest that there is a possibility that a homologue of Pf EBA-165 takes part in the ookinete recognition and invasion process of the mosquito midgut by plasmodia ookinetes.
Assuntos
Anopheles/parasitologia , Antígenos de Protozoários/metabolismo , Mucosa Intestinal/metabolismo , Mucosa Intestinal/parasitologia , Plasmodium berghei/crescimento & desenvolvimento , Animais , Antígenos de Protozoários/análise , Biotinilação , Western Blotting , Cromatografia de Afinidade , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Eritrócitos/metabolismo , Eritrócitos/parasitologia , Feminino , Interações Hospedeiro-Parasita/fisiologia , Ligantes , Espectrometria de Massas , Camundongos , Camundongos Endogâmicos , Plasmodium berghei/patogenicidade , VirulênciaRESUMO
Entomopathogenic nematodes (EPNs) are efficient insect parasites, that are known for their mutualistic relationship with entomopathogenic bacteria and their use in biocontrol. EPNs produce bioactive molecules referred to as excreted/secreted products (ESPs), which have come to the forefront in recent years because of their role in the process of host invasion and the modulation of its immune response. In the present study, we confirmed the production of ESPs in the EPN Heterorhabditis bacteriophora, and investigated their role in the modulation of the phenoloxidase cascade, one of the key components of the insect immune system. ESPs were isolated from 14- and 21-day-old infective juveniles of H. bacteriophora, which were found to be more virulent than newly emerged nematodes, as was confirmed by mortality assays using Galleria mellonella larvae. The isolated ESPs were further purified and screened for the phenoloxidase-inhibiting activity. In these products, a 38 kDa fraction of peptides was identified as the main candidate source of phenoloxidase-inhibiting compounds. This fraction was further analyzed by mass spectrometry and the de novo sequencing approach. Six peptide sequences were identified in this active ESP fraction, including proteins involved in ubiquitination and the regulation of a Toll pathway, for which a role in the regulation of insect immune response has been proposed in previous studies.
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A cDNA encoding elastase was isolated from Steinernema carpocapsae by suppression subtractive hybridization and rapid amplification of 5' cDNA ends. The predicted protein contained a 19-aa signal peptide, a 44-aa N-terminal propeptide, and a 264-aa mature protein with a predicted molecular mass of 28,949 Da and a theoretical pI of 8.88. BLAST analysis showed 27-35% amino acid sequence identity to serine proteases from insects, mammals, fish and other organisms. The Sc-ela gene contains three exons and two introns with at least two copies in the S. carpocapsae genome. Expression analysis indicated that the Sc-ela gene was upregulated during the initial parasitic stage. Sequence comparison and evolutionary marker analysis revealed that Sc-ELA was a member of the elastase serine protease family with potential degradative, developmental and fibrinolytic activities. Homology modeling showed that Sc-ELA adopts a two beta-barrel fold typical of trypsin-like serine proteases, and phylogenetic analysis indicates that Sc-ELA branched off early during elastase evolution.
Assuntos
Regulação Enzimológica da Expressão Gênica , Rabditídios/genética , Serina Proteases/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Southern Blotting , Clonagem Molecular , DNA Complementar/química , Interações Hospedeiro-Parasita , Dados de Sequência Molecular , Controle Biológico de Vetores , Filogenia , RNA de Helmintos/genética , RNA de Helmintos/isolamento & purificação , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rabditídios/classificação , Rabditídios/enzimologia , Alinhamento de Sequência , Serina Proteases/biossíntese , Serina Proteases/química , Especificidade por SubstratoRESUMO
Marine bioadhesives perform in ways that manmade products simply cannot match, especially in wet environments. Despite their technological potential, bioadhesive molecular mechanisms are still largely understudied, and sea urchin adhesion is no exception. These animals inhabit wave-swept shores, relying on specialized adhesive organs, tube feet, composed by an adhesive disc and a motile stem. The disc encloses a duo-gland adhesive system, producing adhesive and deadhesive secretions for strong reversible substratum attachment. The disclosure of sea urchin Paracentrotus lividus tube foot disc proteome led to the identification of a secreted adhesion protein, Nectin, never before reported in adult adhesive organs but, that given its adhesive function in eggs/embryos, was pointed out as a putative substratum adhesive protein in adults. To further understand Nectin involvement in sea urchin adhesion, Nectin cDNA was amplified for the first time from P. lividus adhesive organs, showing that not only the known Nectin mRNA, called Nectin-1 (GenBank AJ578435), is expressed in the adults tube feet but also a new mRNA sequence, called Nectin-2 (GenBank KT351732), differing in 15 missense nucleotide substitutions. Nectin genomic DNA was also obtained for the first time, indicating that both Nectin-1 and Nectin-2 derive from a single gene. In addition, expression analysis showed that both Nectins are overexpressed in tube feet discs, its expression being significantly higher in tube feet discs from sea urchins just after collection from the field relative to sea urchin from aquarium. These data further advocate for Nectin involvement in sea urchin reversible adhesion, suggesting that its expression might be regulated according to the hydrodynamic conditions.
Assuntos
Processamento Alternativo , Moléculas de Adesão Celular/genética , Mecanotransdução Celular/genética , Paracentrotus/genética , Proteoma/genética , Adesividade , Adesivos/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Moléculas de Adesão Celular/metabolismo , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Nectinas , Especificidade de Órgãos , Paracentrotus/química , Paracentrotus/metabolismo , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteoma/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Steinernemacarpocapsae is a nematode pathogenic in a wide variety of insect species. The great pathogenicity of this nematode has been ascribed to its ability to overcome the host immune response; however, little is known about the mechanisms involved in this process. The analysis of an expressed sequence tags (EST) library in the nematode during the infective phase was performed and a highly abundant contig homologous to serine protease inhibitors was identified. In this work, we show that this contig is part of a 641-bp cDNA that encodes a BPTI-Kunitz family inhibitor (Sc-KU-4), which is up-regulated in the parasite during invasion and installation. Recombinant Sc-KU-4 protein was produced in Escherichia coli and shown to inhibit chymotrypsin and elastase activities in a dose-dependent manner by a competitive mechanism with Ki values of 1.8 nM and 2.6 nM, respectively. Sc-KU-4 also inhibited trypsin and thrombin activities to a lesser extent. Studies of the mode of action of Sc-KU-4 and its effects on insect defenses suggest that although Sc-KU-4 did not inhibit the activation of hemocytes or the formation of clotting fibers, it did inhibit hemocyte aggregation and the entrapment of foreign particles by fibers. Moreover, Sc-KU-4 avoided encapsulation and the deposition of clotting materials, which usually occurs in response to foreign particles. We show by protein-protein interaction that Sc-KU-4 targets recognition proteins of insect immune system such as masquerade-like and serine protease-like homologs. The interaction of Sc-KU-4 with these proteins explains the ability of the nematode to overcome host reactions and its large pathogenic spectrum, once these immune proteins are well conserved in insects. The discovery of this inhibitor targeting insect recognition proteins opens new avenues for the development of S. carpocapsae as a biological control agent and provides a new tool to study host-pathogen interactions.
Assuntos
Aprotinina/metabolismo , Etiquetas de Sequências Expressas/metabolismo , Evasão da Resposta Imune/fisiologia , Modelos Moleculares , Nematoides/patogenicidade , Conformação Proteica , Animais , Aprotinina/genética , Western Blotting , Mapeamento de Sequências Contíguas , Primers do DNA/genética , Escherichia coli , Evolução Molecular , Perfilação da Expressão Gênica , Evasão da Resposta Imune/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Steinernema carpocapsae is an entomopathogenic nematode widely used for the control of insect pests due to its virulence, which is mainly attributed to the ability the parasitic stage has to overcome insect defences. To identify the mechanisms underlying such a characteristic, we studied a novel serpin-like inhibitor (sc-srp-6) that was detected in a transcriptome analysis. Recombinant Sc-SRP-6 produced in Escherichia coli had a native fold of serpins belonging to the α-1-peptidase family and exhibited inhibitory activity against trypsin and α-chymotrypsin with Ki of 0.42 × 10(-7) M and 1.22 × 10(-7) M, respectively. Functional analysis revealed that Sc-SRP-6 inhibits insect digestive enzymes, thus preventing the hydrolysis of ingested particles. Moreover, Sc-SRP-6 impaired the formation of hard clots at the injury site, a major insect defence mechanism against invasive pathogens. Sc-SRP-6 does not prevent the formation of clot fibres and the activation of prophenoloxidases but impairs the incorporation of the melanin into the clot. Binding assays showed a complex formation between Sc-SRP-6 and three proteins in the hemolymph of lepidopteran required for clotting, apolipophorin, hexamerin and trypsin-like, although the catalytic inhibition occurred exclusively in trypsin-like. This data allowed the conclusion that Sc-SRP-6 promotes nematode virulence by inhibiting insect gut juices and by impairing immune clot reaction.
Assuntos
Proteínas de Helminto/metabolismo , Insetos/parasitologia , Rabditídios/metabolismo , Serpinas/metabolismo , Animais , Proteínas de Helminto/genética , Hemolinfa/efeitos dos fármacos , Hemolinfa/metabolismo , Proteínas de Insetos/metabolismo , Insetos/metabolismo , Rabditídios/patogenicidade , Serpinas/genéticaRESUMO
Bacillus mycoides S122C was identified as carboxymethyl cellulase (CMcellulase)-producing bacteria from the Azorean Bacillus collection (Lab collection), which was isolated from local soil samples. The bacteria was identified by 16S rRNA sequence and designated as B. mycoides S122C. NCBI blast analysis showed that the B. mycoides S122C 16S rRNA sequence has high identity compared to other B. mycoides strains. CMcellulase was purified from the culture filtrates using anion-exchange chromatography. After mono-Q purification, the protein folds and recovery were 13.7 and 0.76 %, respectively. SDS-PAGE analysis showed that the molecular weight of the purified CMcellulase protein was estimated to be about 62 kDa and that it was composed of a single subunit. MALDI-MS/MS analysis yielded each four peptides of the purified protein; it has identity to other cellulases. The purified CMcellulase showed high activity with CMcellulose followed by ß-glucan as a substrate. Optimum temperature and pH for the purified CMcellulase activity were found to be at 50 °C and pH 7.0, respectively. The purified CMcellulase was stable with about 60 % activity in broad pH ranges from 5 to 10 and temperature of 40 to 60 °C. However, purified CMcellulase was stable at about 70 % at 70 °C and also stable overall at 78 % for surfactants. CMcellulase activity was inhibited by ions such as HgCl(2), followed by CuSo(4), FeCl(2), and MnCl(2), while CoCl(2) activated CMcellulase activity. The purified CMcellulase activity was strongly inhibited by EDTA.
Assuntos
Bacillus/enzimologia , Celulase/química , Celulase/metabolismo , Temperatura , Bacillus/genética , Bacillus/metabolismo , Biomassa , Celulase/biossíntese , Celulase/isolamento & purificação , Celulose/metabolismo , Inibidores Enzimáticos/farmacologia , Estabilidade Enzimática/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Hidrólise , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Análise de Sequência de RNA , Especificidade por Substrato , Tensoativos/farmacologiaRESUMO
Steinernema carpocapsae is an insect parasitic nematode associated with the bacterium Xenorhabdus nematophila. During invasion, this nematode is able to express many proteases, including aspartic proteases. Genes encoding these aspartic proteases have been identified in the EST, and aspartic protease has been found in excretory-secretory products. The total protease was shown to digest blood hemoglobin in a zymogram gel. When the protein was partially purified by pepstatin affinity chromatography, it was observed to have high activity against both hemoglobin and the synthetic substrate Phe-Ala-Ala-Phe-(4NO(2))-Phe-Val-Leu (4-pyridylmethyl) ester. The protein was confirmed by mass spectrometry and was found to be encoded by the gene sc-asp113. A cDNA encoding aspartic protease was cloned based on the EST fragment, which was constructed in our lab. The full-length cDNA of Sc-ASP113 consists of 1257 nucleotides encoding a protein with multiple domains, including a signal peptide (aa 1-15), a propeptide region (aa 16-45), and a typical catalytic aspartic domain (aa 68-416). The cleavage site of the signal peptide is predicted to be between Ala15 and Ala16. The putative 418 amino acid residues have a calculated molecular mass of 44,742Da and a theoretical pI of 5.14. BLAST analysis showed 33-56% amino acid sequence identity to aspartic proteases from parasitic and free living nematodes. Expression analysis showed that the sc-asp113 gene was up-regulated during the initial parasitic stage, especially during L3 inside the gut. In vitro, we showed that treatment with insect homogenate for 6h is sufficient to induce the expression of this protease in treated infective juveniles. Sequence comparison and evolutionary analysis revealed that Sc-ASP113 is a member of the aspartic protease family with the potential for tissue degradation. Phylogenetic analysis indicates that Sc-ASP113 branched between Haemonchus contortus and Steinernema feltiae proteases. Homology modeling showed that Sc-ASP113 adopts a typical aspartic protease structure. The up-regulation of Sc-ASP113 expression indicates that this protease could play a role in the parasitic process. To facilitate the exploration of this protease as a virulence factor, here we describe the purification of the protease and its molecular characterization in S. carpocapsae.
Assuntos
Ácido Aspártico Proteases/isolamento & purificação , Proteínas de Helminto/isolamento & purificação , Nematoides/enzimologia , Nematoides/genética , Animais , Ácido Aspártico Proteases/genética , Ácido Aspártico Proteases/metabolismo , Domínio Catalítico , Cromatografia de Afinidade/métodos , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/metabolismo , Etiquetas de Sequências Expressas , Feminino , Regulação da Expressão Gênica , Genes de Helmintos , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Hemoglobinas , Interações Hospedeiro-Parasita , Insetos/parasitologia , Masculino , Espectrometria de Massas , Nematoides/patogenicidade , Infecções por Nematoides/parasitologia , Filogenia , Sinais Direcionadores de Proteínas , Fatores de Tempo , Regulação para CimaRESUMO
Steinernema carpocapsae is a parasitic nematode that is high virulent to insects. The parasitic juvenile reaches the insect haemocoelium by passing through mid-gut barriers and develops there. During invasion, the nematode was predicted to express a large set of proteases, including metalloproteases, one of which was sequenced and expressed in this work. A 1583-nucleotide cDNA encoding a putative metalloprotease containing a 28-aa signal peptide, a 79-aa propeptide and a 311-aa mature protease with a predicted molecular mass of 35.2 kDa and a theoretical pI of 5.9 was cloned from the parasitic stage of the nematode. Sequence analyses predicted signature sequences of the astacin metalloprotease family, an astacin domain, a zinc-binding motif and a methionine turn motif; therefore, this protein was identified as an astacin and designated as Sc-AST. The astacin domain of Sc-AST has an amino acid sequence homology of 46% to prototypical astacin from Astacus astacus and 82% to Caenorhabditis elegans NAS-8. Like NAS-8 of C. elegans, Sc-AST has a C-terminal ShK toxin domain. Recombinant Sc-AST was produced in an Escherichia coli system and was purified by affinity chromatography. Maldi-MS/MS analysis of purified recombinant protein matched the Sc-AST sequence with a significance score of 499. Sc-AST was produced in the correct folding conformation, showed activities against gelatin and azocasein substrates and was inhibited by divalent metal-chelating agents. Sc-AST presented an optimum pH of 7.5 and temperature of 37°C and K(m), V(max) and k(cat) values of 1.86 mM, 0.281 µM/min and 27.9 s(-1), respectively. Expression analyses indicated that Sc-AST is up-regulated in the parasitic stage and is strongly induced in vitro by insect tissues, thus suggesting that it plays a role in the parasitic process.
Assuntos
Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Metaloendopeptidases , Mariposas/parasitologia , Rabditídios/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA Complementar , Proteínas de Helminto/química , Proteínas de Helminto/genética , Proteínas de Helminto/metabolismo , Metaloendopeptidases/química , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rabditídios/genética , Rabditídios/crescimento & desenvolvimento , Alinhamento de SequênciaRESUMO
A chymotrypsin serine protease (designated Sc-CHYM) was purified by gel filtration and anion-exchange chromatography from excretory-secretory products of parasitic stage Steinernema carpocapsae. The purified protease had an apparent molecular mass of 30kDa and displayed a pI of 5.9. This protease demonstrated high activity against the chymotrypsin-specific substrate N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide and was highly sensitive to the inhibitor aprotinin. This protease digested the chromogenic substrate N-Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide with K(m), V(max) and k(cat) values of 409microM/min, 0.389microM/min and 24.9s(-1), respectively. The protease was most active at pH 8.0 and 35 degrees C, and its proteolytic activity was almost completely reduced after incubation at 75 degrees C for 30min. In vitro, this enzyme suppressed prophenoloxidase activity. In vivo, demonstration of encapsulation and melanization by purified chymotrypsin imbibed beads showed it could prevent hemocyte encapsulation and melanization by 12 and 24h, respectively. Sequence comparison and evolutionary marker analysis showed that the putative protein was a chymotrypsin-like protease with potential degradative, developmental and fibrinolytic functions. Expression pattern analysis revealed that the gene expression of Sc-CHYM was up-regulated in the parasitic stage. Sc-CHYM was clustered with several insect chymotrypsins and formed an ancestral branch in the phylogenetic tree, suggesting that Sc-CHYM branched off at an early stage of cluster divergence. The results of this study suggest that Sc-CHYM is a new member of the chymotrypsin serine protease family and that it might act as a virulence factor in host-parasite interactions.
Assuntos
Quimotripsina/genética , Regulação Enzimológica da Expressão Gênica/genética , Interações Hospedeiro-Parasita/genética , Insetos/parasitologia , Nematoides/genética , Serina Proteases/genética , Animais , Catecol Oxidase/antagonistas & inibidores , Quimotripsina/metabolismo , Precursores Enzimáticos/antagonistas & inibidores , Dados de Sequência Molecular , Nematoides/metabolismo , Controle Biológico de Vetores , Serina Proteases/biossínteseRESUMO
Steinernema carpocapsae is an insect parasitic nematode able to parasitise and kill the host within 48 h. Secreted products (ESP) of the parasitic stage of a virulent strain contain higher amounts of proteolytic activity than a low virulence strain, suggesting proteases are involved in virulence. From the ESP we purified a protein (Sc-SP-3) with a M(r) of 30 kDa and a pI of 7 that cleaved the synthetic substrate N-succinyl-Ala-Ala-Pro-Phe-pNA and was inhibited by phenylmethanesulfonyl fluoride, benzamidine and chymostatin, thus indicating that it belongs to the chymotrypsin-like serine protease family. Sc-SP-3 has a V(max) of 0.3 mM min(-1)ml(-1) and K(m) of 6.6 x 10(-4)M, with maximum activity at pH 8 and 40 degrees C. The full-length cDNA was obtained using degenerate oligonucleotides for serine proteases. This open reading frame encodes a preproprotein containing a putative signal peptide composed of 16 amino acid residues, a prodomain of 40 residues and a mature protease domain of 261 residues, including the catalytic triad His/Asp/Ser characteristic of trypsin-like serine proteases. The N-terminal sequence and the peptide masses fingerprint obtained by MALDI-TOF-MS for the purified protein matched the cDNA. Gene expression analysis by quantitative real-time-PCR showed that this gene is expressed only during the parasitic stage and that pre-invasive nematodes inside the mid-gut expressed higher amounts of Sc-SP-3 than those that already enter the haemocoel. Sc-SP-3 caused histolysis in the insect mid-gut. In vitro assays demonstrated that Sc-SP-3 digested extracellular proteins and induced apoptosis in Sf9 insect cells, thus suggesting Sc-SP-3 is a multifunctional chymotrypsin-like protease involved in pathogenesis.