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

Gonzalo Ernesto González-Páez,Raúl Argüello-García,Fernando Alba-Hurtado,Carlos Gerardo García-Tovar

doi:10.1155/2014/418708

Abstract

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.

Highlights

Toxocariasis is caused in dog and human hosts by infection with the larvae of the ascarid worm Toxocara canis
When gels were copolymerized with gelatin, proteolytic activities developed at the three different pH values employed (5.5, 7.6, and 9.0; Figure 1, lanes 2–4, resp.) and only the ∼400 and 120 kDa bands displayed a significant activity that was similar at the different pH values evaluated
In order to determine the type of proteinases contained in Toxocara excretory-secretory products (TES), samples were subjected to electrophoretic separation using representative substrate and pH 5 (7.6) conditions and proteolytic activity was developed by previous treatment of gels with proteinase inhibitors with selectivity for cysteine, serine, aspartyl, and metalloproteinases

Summary

Introduction

Toxocariasis is caused in dog (definitive) and human (paratenic) hosts by infection with the larvae of the ascarid worm Toxocara canis. Puppies infected at the intestine with the adult stage of T. canis shed in feces large number of infective eggs into the environment. When these are accidentally ingested by a patient, the infective larva (L2) hatches, penetrates the intestinal wall, and through the circulatory system targets various organs [1]. Larvae migrating throughout bodily fluids (i.e. blood) and somatic organs shed large amounts of lipids and immunogenic glycoproteins known together as Toxocara excretory-secretory products (TES) which have been proposed to serve a strategy to BioMed Research International escape the immune attack of the host [14]. The larva migrans status in toxocariasis is associated with worms lacking anchoring apparatus (oral appendages, stylets, hooks, or scolex); other biological functions have to be considered for TES in the completion of Toxocara life cycle

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