Identification of the Secreted Neutral Proteases from Anisakis simplex

Abstract

Ingestion of larval nematodes (family: Anisakidae) can cause the human disease known as anisakiasis. After ingestion, Anisakis larvae can be invasive, penetrating host stomach or intestinal wall. Observation of larvae penetrating the tissue layers of human stomach in vitro by SEM showed tunnels and burrows were formed in the mucosa and submucosa. Based on these observations, we hypothesized that secreted proteases may be involved in the degradation of host tissue macromolecules to allow tunnel formation. Using a model of connective tissue extracellular matrix (ECM), we found that as few as 5 Anisakis simplex larvae could degrade approximately 25% of the ECM in a 16-mm culture well in 24 hr. Further characterization of the secreted proteases using synthetic peptide substrates and inhibitors revealed that there were 2 classes of proteases present: a metallo aminopeptidase and a trypsinlike serine protease. Extracts of Anisakis larvae contained a 25-kDa protease that was recognized by rabbit anti-rat trypsin antibody on western blots. This suggests that there is structural as well as functional similarity between the Anisakis trypsin and vertebrate trypsins.

The helminth community ofGeophagus proximus(Perciformes: Cichlidae) from a tributary of the Paraná River, Ilha Solteira Reservoir, São Paulo State, Brazil

“…Humans acquire this infection by eating raw food dishes such as sushi, sashimi, ceviche or undercooked fish and squid dishes (Sakanari & McKerrow, 1990). This disease occurs mainly in countries whose populations have the habit of eating raw fish, such as those belonging to the Asian continent (Asami et al, 1965;Sohn & Seol, 1994).…”

Activity of selected hydrolases in excretion-secretion products and homogenates from L 3 and L 4 larvae of Anisakis simplex (Nematoda: Anisakidae) parasitising herring

“…According to numerous authors, aminopeptidases are important as activators of hormone-and enzyme precursors during the hatching and moulting of larvae (Rhoads et al 1997). Leucine aminopeptidase was found to be active in numerous parasites and also during hatching and moulting of their larvae (Rogers 1982, Sakanari and McKerrow 1990, Niemczuk 1993, Rhoads et al 1997. Aminopeptidase activity was detected in a medium containing Ascaris suum larvae when these were moulting from L 3 to L 4 (Rhoads et al 1997).…”

“…Most of the available literature on ES products in A. simplex stage-3 larvae focuses primarily on protease activity (Matthews 1982, 1984, Kennedy et al 1988, Sakanari and McKerrow 1990, Morris and Sakanari 1994. Two proteases: trypsin-like serinpeptidase and aminopeptidase were found to be active.…”

Collagenolytic activity related to metalloproteases (and serine proteases) in the fish parasite Hysterothylacium aduncum (Nematoda: Anisakidae)

“…Significant differences were found between parasites cultivated with and without pepsin (2-way ANOVA, p < 0.001), enzymatic activity being greatest in the latter, although the activity profiles were similar throughout. In other nematodes from the same family, such as Anisakis simplex, serine and metalloprotease activity have been demonstrated in their excretion-secretion (ES) products, sufficient to degrade the collagen in the connective tissue of an extracellular matrix (Sakanari & McKerrow 1990). The connective tissue of the gastrointestinal tract contains collagen Type I, the substrate used in the present study.…”