Tissue-specific distribution of serine/threonine protein phosphatase 1 of Toxocara canis.

Serine/threonine protein phosphatase 1 (PP1) is expressed in developing and reproductively active male Toxocara canis. To investigate the tissue-specific expression of PP1 in T. canis, the PP1 protein was expressed in Escherichia coli, and the recombinant protein was used to generate a rabbit polyclonal antiserum. Indirect fluorescence immunohistochemical analysis of adult male T. canis showed that PP1 was expressed in the germ line tissues, primarily in the testis, seminal vesicle, vas deferens, and sperm cells, indicating the potential roles of PP1 in spermatogenesis. What's more, structural predictions of PP1 in T. canis were performed. The predictions of the structure indicated that PP1 may be a potential target for antihelmintic drugs. This is the first report of the tissue distributions and structural prediction of PP1 in T. canis, which might lead to the development of novel, innovative strategies for controlling T. canis infestations.

Proteinases in excretory-secretory products of Toxocara canis second-stage larvae: zymography and modeling insights.

Components released in excretory-secretory products of Toxocara canis larvae (TES) include phosphatidylethanolamine-binding proteins (TES26), mucins (TES120, MUC2-5), and C-type lectins (TES32, TES70) and their biochemical, immunological, and diagnostic properties have been extensively studied albeit proteinase activities towards physiological substrates are almost unknown. Proteolytic activities in TES samples were first analyzed by gel electrophoresis with gelatin as substrate. Major activities of ~400, 120, and 32 kDa in TES were relatively similar over a broad pH range (5.5-9.0) and all these were of the serine-type as leupeptin abolished gelatinolysis. Further, the ~400 kDa component degraded all physiological substrates tested (laminin, fibronectin, albumin, and goat IgG) and the 120 kDa component degraded albumin and goat IgG while proteinases of lower MW (45, 32, and 26 kDa) only degraded laminin and fibronectin, preferentially at alkaline pH (9.0). By protein modeling approaches using the known sequences of TES components, only TES26 and MUC4 displayed folding patterns significantly related to reference serine proteinases. These data suggest that most of serine proteinase activities secreted in vitro by infective larvae of T. canis have intriguing nature but otherwise help the parasite to affect multiple components of somatic organs and bodily fluids within the infected host.

Molecular characterization and functional analysis of serine/threonine protein phosphatase of Toxocara canis.

Toxocara canis (T. canis) is a widely prevalent zoonotic parasite that infects a wide range of mammalian hosts, including humans. We generated the full-length complementary DNA (cDNA) of the serine/threonine phosphatase gene of T. canis (Tc stp) using 5' rapid amplification of the cDNA ends. The 1192-bp sequence contained a continuous 942-nucleotide open reading frame, encoding a 313-amino-acid polypeptide. The Tc STP polypeptide shares a high level of amino-acid sequence identity with the predicted STPs of Loa loa (89%), Brugia malayi (86%), Oesophagostomum columbianum (76%), and Oesophagostomumdentatum (76%). The Tc STP contains GDXHG, GDXVDRG, GNHE motifs, which are characteristic of members of the phosphoprotein phosphatase family. Our quantitative real-time polymerase chain reaction analysis showed that the Tc STP was expressed in six different tissues in the adult male, with high-level expression in the spermary, vas deferens, and musculature, but was not expressed in the adult female, suggesting that Tc STP might be involved in spermatogenesis and mating behavior. Thus, STP might represent a potential molecular target for controlling T. canis reproduction.

Toxocara canis: molecular cloning, characterization, expression and comparison of the kinetics of cDNA-derived arginine kinase.

Arginine kinase (AK) is a member of a highly conserved family of phosphagen kinases. We determined the cDNA sequence of Toxocara canis AK, cloned it in pMAL plasmid and expressed it in Escherichia coli as a fusion protein with maltose-binding protein. The protein has a theoretical molecular mass of 45,376 Da and an estimated isoelectric point (pI) of 8.38. Alignment of the cDNA-derived amino acid sequence of T. canis AK with other phosphagen kinase sequences showed high amino acid identity with other nematode AKs, and phylogenetic analysis placed it as a distinct branch within a nematode AK cluster. Analysis of the N-terminus sequence of T. canis AK revealed the presence of a signal targeting peptide presumably targeting this protein to cytosol or endoplasmic reticulum (ER). T. canis AK showed high activity for l-arginine. The kinetic constants (K(m) = 0.12 mM, K(cat) = 29.18, and K(d) = 0.23 mM) and V(max) (43.76 micromolPi/min/mg protein) of T. canis recombinant-AK were determined for the forward reaction. It also exhibited a synergism for substrate binding (K(d)(Arg)/K(m)(Arg)=1.96). Comparison of K(cat)/K(m)(Arg) values in various arginine kinases indicates that T. canis AK has a high catalytic efficiency (248.19s(-1)mM(-1)). The present study contains the first description of arginine kinase in a zoonotic nematode. The determination of T. canis AK and its phosphagen biosynthetic pathway, which is completely different from those in mammalian host tissues, suggests this enzyme as a possible novel chemotherapy target for VLM syndrome in humans.

Toxocara canis: proteinases in perivitelline fluid from hatching eggs.

Developing larvae of Toxocara canis may secrete several kinds of enzymes within the egg perivitelline fluid (EPF) prior to and during hatching. In particular, proteinases in EPF could play a role in larval emergence within the host gastrointestinal lumen but its presence and nature is unknown. In this work, proteolytic activities in hatching fluid of T. canis were identified and analysed by substrate gel electrophoresis at different pH values and by using type specific protease inhibitors. Three bands of 91, 68 and 38 kDa showed gelatinolytic activity and all proteinase activity from EPF was of the aspartic-type since it was inhibited by pepstatin A. Interestingly, a significantly higher proteolytic activity was observed at acidic pH (< or =5.5). These data suggest that T. canis developmentally secretes and accumulates in EPF aspartic proteinases with a pH-dependent activity that might help the parasite to take advantage of conditions in the host gastrointestinal microenvironment where egg hatching is induced and executed.

Enhanced inducible nitric oxide synthase expression and nitrotyrosine accumulation in experimental granulomatous hepatitis caused by Toxocara canis in mice.

The involvement of inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT) in pathogenesis of toxocaral granulomatous hepatitis (TGH) in a murine host was quantitatively determined by biochemical, parasitological, pathological, and immunohistochemical assessments in a 42-week investigation. Mice were sacrificed for serum collection and histological processing as well as acid-pepsin digestion of the liver in a larval recovery study. Significantly increased levels of total serum NO were found in the trial, indirectly suggesting iNOS activation in the liver. iNOS reactivity was predominantly observed in infiltrating leucocytes in lesions and normal and apocrine-like cholangiocytes; in contrast, hepatocytes and multinucleated giant cells showed negative cytoplasmic staining in TGH. Strong iNOS-like reactivity was also detected on the body wall of larvae. The locations of NT reactivity were nearly identical to those of iNOS expression; infiltrating leucocytes or cholangiocytes stained for iNOS were also stained for NT in TGH. Enhanced iNOS expression, but not invading larvae (r = 0.256, P = 0.211), seemed to play a certain role in pathological damage in TGH due to a significant correlation between iNOS expression and serum alanine aminotransferase (ALT) levels (r =0.593, P = 0.021) in the trial. Our present results indicate a potential therapeutic strategy for treatment of GH caused by other nematodes through manipulation of iNOS expression.

Mucin-type O-glycosylation in helminth parasites from major taxonomic groups: evidence for widespread distribution of the Tn antigen (GalNAc-Ser/Thr) and identification of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferase activity.

This article focuses on the initiation pathway of mucin-type O-glycosylation in helminth parasites. The presence of the GalNAc-O-Ser/Thr structure, also known as Tn antigen, a truncated determinant related to aberrant glycosylation in mammal cells, and the activity of the UDP-GalNAc:polypeptide N-acetyl-galactosaminyltransferase (ppGaNTase), the enzyme responsible for its synthesis, were studied in species from major taxonomic groups. Tn reactivity was determined in extracts from Taenia hydatigena, Mesocestoides corti, Fasciola hepatica, Nippostrongylus brasiliensis, and Toxocara canis using the monoclonal antibody 83D4. The Tn determinant was revealed in all preparations, and multiple patterns of Tn-bearing glycoproteins were observed by immunoblotting. Additionally, the first evidence that helminth parasites express ppGaNTase activity was obtained. This enzyme was studied in extracts from Echinococcus granulosus, F. hepatica, and T. canis by measuring the incorporation of UDP-(3H)GalNAc to both deglycosylated ovine syalomucin (dOSM) and synthetic peptide sequences derived from tandem repeats of human mucins. Whereas significant levels of ppGaNTase activity were detected in all the extracts when dOSM was used as a multisite acceptor, it was only observed in F. hepatica and E. granulosus extracts when mucin-derived peptides were used, suggesting that T. canis ppGaNTase enzyme(s) may represent a member of the gene family with a more restricted specificity for worm O-glycosylation motifs. The widespread expression of Tn antigen, capable of evoking both humoral and cellular immunity, strongly suggests that simple mucin-type O-glycosylation does not constitute an aberrant phenomenon in helminth parasites.

In vivo inhibition of inducible nitric oxide synthase decreases lung injury induced by Toxocara canis in experimentally infected rats.

The direct effect of nitric oxide (NO) on the viability of Toxocara canis larvae was studied. We observed that the nitric oxide donors, SIN-1 and SNOG, exert no cytotoxic effect on the in vitro viability of T. canis larvae. In addition, we developed a model in rats to elucidate the role of NO during T. canis infection. We evaluated different indicators in four experimental groups: morphological parameters, the total number cells and cell types recovered, nitrite and protein concentration, lactate dehydrogenase and alkaline phosphatase enzymatic activity in the bronchoalveolar lavage fluid, lung index and detection of anti-T. canis specific antibodies. We observed significant differences between non-infected and infected groups. The infected animals treated with the inducible nitric oxide synthase (iNOS) inhibitor aminoguanidine were less damaged than infected, non-treated animals. Our results suggest that the in vivo inhibition of the synthesis of NO triggered by iNOS diminishes the deleterious effects of the parasite upon the host, especially the vascular alterations in the lungs. We could show that in vivo production of NO induced by infection with T. canis results in direct host damage. Thus, this induction may constitute an evasion/adaptation mechanism of the parasite.

Characterisation of Tc-cpl-1, a cathepsin L-like cysteine protease from Toxocara canis infective larvae.

Cysteine proteases play vital biological roles in both intracellular and extracellular environments. A cysteine protease migrating at 30 kDa was identified in somatic extracts of Toxocara canis larvae (TEX), by its binding to the biotinylated inhibitor Phe-Ala-CH2F. TEX proteases readily cleaved the cathepsin L- and B-specific peptide substrate Z-Phe-Arg-AMC and to a lesser extent, the cathepsin B-specific peptide Z-Arg-Arg-AMC. Excretory/secretory (TES) products of T. canis larvae did not cleave either substrate. Partial sequence encoding the 5' end of a cysteine protease cDNA from infective T. canis larvae was then obtained from an expressed sequence tag (EST) project. The entire cDNA (termed Tc-cpl-1) was subsequently sequenced and found to encode a preproenzyme similar to cathepsin L-like proteases (identities between 36 and 69%), the closest homologues being two predicted proteins from Caenorhabditis elegans cosmids, a cathepsin L-like enzyme from Brugia pahangi and a range of parasite and plant papain-like proteases. Sequence alignment with homologues of known secondary structure indicated several charged residues in the S1 and S2 subsites involved in determining substrate specificity. Some of these are shared with human cathepsin B, including Glu 205 (papain numbering), known to permit cleavage of Arg-Arg peptide bonds. The recombinant protease (rTc-CPL-1) was expressed in bacteria for immunisation of mice and the subsequent antiserum shown to specifically react with the 30 kDa native protease in TEX. Sera from mice infected with the parasite also contained antibodies to rTc-CPL-1 as did sera from nine patients with proven toxocariasis; control sera did not. Larger scale studies are underway to investigate the efficacy of rTc-CPL-1 as a diagnostic antigen for human toxocariasis, the current test for which relies on whole excretory/secretory antigens of cultured parasites.