Pepsin-like aspartic protease (Sc-ASP155) cloning, molecular characterization and gene expression analysis in developmental stages of nematode Steinernema carpocapsae.

Steinernema carpocapsae is an insect parasitic nematode associated with the bacterium Xenorhabdus nematophila. These symbiotic complexes are virulent against the insect host. Many protease genes were shown previously to be induced during parasitism, including one predicted to encode an aspartic protease, which was cloned and analyzed in this study. A cDNA encoding Sc-ASP155 was cloned based on the EST fragment. The full-length cDNA of Sc-ASP155 consists of 955 nucleotides with multiple domains, including a signal peptide (aa1-15), a pro-peptide region (aa16-45), and a typical catalytic aspartic domain (aa71-230). The putative 230 amino acid residues have a calculated molecular mass of 23,812Da and a theoretical pI of 5.01. Sc-ASP155 blastp analysis showed 40-62% amino acid sequence identity to aspartic proteases from parasitic and free-living nematodes. Expression analysis showed that the sc-asp155 gene was up-regulated during the initial parasitic stage, especially in L3 gut and 6h induced nematodes. Sequence comparison revealed that Sc-ASP155 was a member of an aspartic protease family and phylogenetic analysis indicated that Sc-ASP155 was clustered with Sc-ASP113. In situ hybridization showed that sc-asp155 was expressed in subventral cells. Additionally, we determined that sc-asp155 is a single-copy gene in S. carpocapsae. Homology modeling showed that Sc-ASP155 adopts a typical aspartic protease structure. The up-regulated Sc-ASP155 expression revealed that this protease could play a role in the parasitic process. In this study, we have cloned the gene and determined the expression of the pepsin-like aspartic protease Sc-ASP155 in S. carpocapsae.

Purification, molecular characterization and gene expression analysis of an aspartic protease (Sc-ASP113) from the nematode Steinernema carpocapsae during the parasitic stage.

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.

Serine protease-mediated host invasion by the parasitic nematode Steinernema carpocapsae.

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.

An apoptosis-inducing serine protease secreted by the entomopathogenic nematode Steinernema carpocapsae.

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.

Identification and expression analysis of the Steinernema carpocapsae elastase-like serine protease gene during the parasitic stage.

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.

Prevalência de dores músculo-esqueléticas em estudantes do ensino médio público do sub-distrito de Santana / Prevalence of musculoskeletal pain among high school students of the Santana district

Esse trabalho teve como objetivos constatar a prevalência de dores músculo-esqueléticas em estudantes do ensino médio. Os sujeitos da pesquisa foram 100 alunos que cursavam o ensino médio, sendo 54 do sexo feminino e 46 do sexo masculino, com idades entre 14 e 21 anos. Foi utilizado um questionário com 10 questões elaboradas pelos autores sobre a localização da dor, intensidade de dor, interferências nas atividades diárias, duração da dor, quando a dor estava presente e prática de atividade física desportiva. As dores que prevaleceram foram coluna vertebral seguida de dor nos joelhos, entre outras. Sobre a intensidade das dores, 57,6% dos participantes consideraram-na moderada, sendo que a minoria considerou a dor forte. A média de interferência das atividades diárias foi de 3,8 em uma escala de 0 a 10, sendo que o grupo feminino foi mais afetado em relação ao grupo masculino. Sobre a duração das dores 73,5% dos indivíduos relatou ter dor de 1 a 7 dias, e apenas 12% apresentou dor com duração maior do que 30 dias. Concluiu-se que as dores músculo-esqueléticas têm grande prevalência na população estudada.

Purification, biochemical and molecular analysis of a chymotrypsin protease with prophenoloxidase suppression activity from the entomopathogenic nematode Steinernema carpocapsae.

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.

Identification, characterization of functional candidate genes for host-parasite interactions in entomopathogenetic nematode Steinernema carpocapsae by suppressive subtractive hybridization.

Identifying parasitism genes encoding proteins secreted from nematodes is the key to understanding the molecular basis of nematode parasitism to insects. In this paper, a cDNA with two introns and three exons encoding a cysteine protease inhibitor was identified by screening a cDNA subtractive library constructed from the nematode, Steinernema carpocapsae, induced by Galleria mellonella hemolymph. The full-length cDNA contains an open reading frame encoding a 139-amino acid protein, designated Sc-cys, with a 19-residue signal peptide. The mature protein was predicted to have a molecular weight of 12,531.59 Da, a pI of 9.44, one disulfide bond, and three conserved domains believed to be important for the inhibition of cysteine proteases. In Basic Local Alignment and Search Tool analyses, the putative protein precursor displayed 26-42% identities to a multitude of cystatins or cystatin-like proteins. Phylogenetic analysis suggested the novel cystatin is likely a new member of the family 2 cystatins. Reverse northern blot, semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR), and real-time RT-PCR analyses showed that the expression level of Sc-cys was upregulated substantially after induction by insect hemolymph. The specific analysis of genes encoding secretory proteins is providing a profile of putative parasitism genes expressed in S. carpocapsae throughout the parasitic cycle.