Nelson Simões

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.

Insect immunity - effects of factors produced by a nematobacterial complex on immunocompetent cells

During in vitro incubations, the nematobacterial complex Steinernema carpocapsae-Xenorhabdus nematophilus produces different factors having toxic activities in vitro towards haemocytes, the insect cells responsible for cellular immune defense reactions. Among others, two effects were evident on haemocyte monolayers; one of them was a cytotoxic activity while the other was an unsticking effect. The factors responsible for cytotoxic activity and unsticking effect, were separated from each other by a single chromatography on anion exchange column. These two effects on haemocytes were lost after heat treatment at 57 degrees C for 1 h and 45 degrees C for 30 min, respectively. Both factors were recovered after dialysis in a 10(4) Da cut off membrane. The cytotoxic activity was susceptible to proteases. Cytotoxic and unsticking factors did not show any lipase or lecithinase activity but the unsticking factor had protease activity. Lipopolysaccharides, purified from the bacteria harvested after incubation of the complex, did not have cytotoxic or unsticking effect on the insect cells in vitro.

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.

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 × 104 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.

Mitochondrial DNA Mutations in Cancer: A Review

As mitochondria participate in fundamental process of the cellular metabolism, recent research has addressed the role of mitochondria, and of mitochondrial DNA (mtDNA), in apoptosis, aging, and complex diseases. The association between mtDNA and cancer has been discussed since the beginning of the last century, and more recently, it has gained attention due to the observation of many somatic mutations in several types of cancers. In this review we describe those germinal mutations that have been associated to cancer, and present a compilation of somatic mutations that have been observed in different cancer tissues, describing relevant characteristics among them in a phylogenetic context. We also summarize the drawbacks and criticisms made towards the studies that report an association between mtDNA mutations and cancer, and discuss the experimental models used to analyse this relationship. Although many reported somatic mutations may actually be the outcome of laboratory artefacts, a considerable number could be authentic and may have a relationship with cancer development. In our compilation, we have observed 271 cancer mutations occurring in conserved positions of mtDNA, 70 of them appearing in more than one tumour. These mutations may be candidates to be used as cancer biomarkers, and deserve further investigation, perhaps through the use of experimental models and by an analysis of tumours of distinct grade to determine if the mutations arose early during tumourigenesis. Experiments with cybrids have been successfully used; however, models are needed in which specific mtDNA variants may be introduced into the same mitochondrial and cellular background.

Increased transcript diversity: novel splicing variants of Machado–Joseph Disease gene (ATXN3)

Machado–Joseph disease (MJD) is a late-onset neurodegenerative disorder that presents clinical heterogeneity not completely explained by its causative mutation. MJD is caused by an expansion of a CAG tract at exon 10 of the ATXN3 gene (14q32.1), which encodes for ataxin-3. The main goal of this study was to analyze the occurrence of alternative splicing at the ATXN3 gene, by sequencing a total of 415 cDNAs clones (from 20 MJD patients and 14 controls). Two novel exons are described for the ATXN3 gene. Fifty-six alternative splicing variants, generated by four types of splicing events, were observed. From those variants, 50 were not previously described, and 26 were only found in MJD patients samples. Most of the variants (85.7%) present frameshift, which leads to the appearance of premature stop codons. Thirty-seven of the observed variants constitute good targets to nonsense-mediated decay, the remaining are likely to be translated into at least 20 different isoforms. The presence of ataxin-3 domains was assessed, and consequences of domain disruption are discussed. The present study demonstrates high variability in the ATXN3 gene transcripts, providing a basis for further investigation on the contribution of alternative splicing to the MJD pathogenic process, as well as to the larger group of the polyglutamine disorders.

Transcripts analysis of the entomopathogenic nematode Steinernema carpocapsae induced in vitro with insect haemolymph

Steinernema carpocapsae is an insect parasitic nematode widely used in pest control programs. The efficacy of this nematode in controlling insects has been found to be related to the pathogenicity of the infective stage. In order to study the parasitic mechanisms exhibited by this parasite, a cDNA library of the induced S. carpocapsae parasitic phase was generated. A total of 2500 clones were sequenced and 2180 high-quality ESTs were obtained from this library. Cluster analysis generated a total of 1592 unique sequences including 1393 singletons. About 63% of the unique sequences had significant hits (e</=1e-05) to the non-redundant protein database. The remaining sequences most likely represent putative novel protein coding genes. Comparative analysis identified 377 homologs in C. elegans, 431 in C. briggsae and 75 in other nematodes. Classification of the predicted proteins revealed involvement in diverse cellular, metabolic and extracellular functions. One hundred and nineteen clusters were predicted to encode putative secreted proteins such as proteases, proteases inhibitors, lectins, saposin-like proteins, acetylcholinesterase, anti-oxidants, and heat-shock proteins, which can possibly have host interactions. This dataset provides a basis for genomic studies towards a better understanding of the events that occur in the parasitic process of this entomopathogenic nematode, including invasion of the insect haemocoelium, adaptations to insect innate immunity and stress responses, and production of virulence factors. The identification of key genes in the parasitic process provides useful tools for the improvement of S. carpocapsae as a biological agent.

INSECT IMMUNITY : THE HAEMOCYTES OF THE ARMYWORM MYTHIMNA UNIPUNCTA (LEPIDOPTERA: NOCTUIDAE) AND THEIR ROLE IN DEFENCE REACTIONS. IN VIVO AND IN VITRO STUDIES

Abstract Haemocytes of Mythimna unipuncta were examined in light and transmission electron microscopy. We describe the different haemocyte types encountered in the sixth instar larvae, the development of the differential haemocyte counts, total haemocyte counts and haemolymph volume. We show the reactions against foreign bodies (encapsulation, phagocytosis) in vivo and compare these reactions under in vitro conditions. These in vitro studies are important to investigate the mode of action of depressive substances purified from parasites or pathogens.

Cloning, characterisation and heterologous expression of an astacin metalloprotease, Sc-AST, from the entomoparasitic nematode Steinernema carpocapsae

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.

Susceptibility of Pseudaletia unipuncta (Lepidoptera: Noctuidae) to Entomopathogenic Nematodes (Rhabditida: Steinernematidae and Heterorhabditidae) Isolated in the Azores: Effect of Nematode Strain and Host Age

Abstract The armyworm, Pseudaletia unipuncta (Haworth), is a serious pest to the Azores’s pastures. In laboratory bioassays we tested the susceptibility of this insect to entomopathogenic nematodes isolated in Azores: Steinernema carpocapsae Az20, Az150, and A48 strains, S. glaseri Az26 strain and Heterorhabditis bacteriophora Az33 strain. The A48, Az20, and Az150 strains caused parasitism rates of 96.6, 90, and 53.3%, and mortality rates of 63.3, 46.6, and 23.3%, respectively, to sixth instar. The Az33 strain caused a parasitism rate of 73.3% and a mortality rate of 40%; whereas, the Az26 strain caused a parasitism rate of 40% and no mortality. A linear response dose—parasitism with a positive regression (r2 = 0.993) was observed in insects exposed to S. carpocapsae Az150 strain. Positive regressions were also observed between mortality and dose rate for S. carpocapsae A48 (r2 = 0.980), Az20 (r2 = 0.956), and Az150 (r2 = 0.963) strains, and H. bacteriophora Az33 strain (r2 = 0.999). Fourth instars were the most susceptible to the A48 strain, followed by the fifth instars, while the sixth instars were the less susceptible, with LD50 values of 26.2, 62.8, and 320.7 infective juveniles, respectively. The lethal time for each of the tested instars was 32.3, 35.5, and 49.2 h, respectively. The invasion rate was 33.5, 28.2, and 40.8 nematodes per treated larvae in the fourth, fifth, and sixth instars, respectively.