Differential cleaving of specific substrates for cathepsin-like activity shows cysteine and serine protease activities and a differential profile between Anisakis simplex s.s. and Anisakis pegreffii, sibling species major etiologic agents of anisakiasis

Abstract

Humans can contract anisakiasis by eating fish or squid containing live larvae of the third stage (L3) of the parasitic nematodes of the genus Anisakis, majorly from A. simplex s.s. and A. pegreffii, sibling species of the A. simplex s.l. complex. Most cases diagnosed molecularly are due to A. simplex s.s., although A. pegreffii has also been identified in human cases. Cathepsins are mostly lysosomal multifunctional cysteine proteases and can participate in the pathogenicity of parasites. Cathepsin B and L activities were investigated in the two sibling species of Anisakis mentioned. L3 and L4 of both species were collected during their in vitro development and cathepsin activity was determined in the range of pH 4.0-8.5, using specific fluorogenic substrates. The activity detected with the substrate Z-FR-AMC was identified as cathepsin L (optimum pH = 5.0, range 4.0-6.0, p<0.001). Activity was highest in L3 freshly collected from fish and decreased during development, which could be related to virulence, invasion of host tissues and/or intracellular digestion. Cathepsin B-like activity was not identified with either of the substrates used (Z-RR-AMC and Z-FR-AMC). With Z-RR-AMC, cleaving activity was detected almost exclusively in L4 of A. simplex s.s. (p<0.05) with optimum pH = 8.0 (range 7.0-8.5). Assays with class-specific protease inhibitors showed this activity was mainly due to serine proteases (up to 90% inhibition with AEBSF), although metalloproteases (up to 40-45% inhibition with 1,10 phenanthroline) and slight cysteine protease activity (<15 % inhibition with E64) were also detected. These results show differential serine protease activity between sibling Anisakis species, regulated by larval development, at least in A. simplex s.s. The higher cathepsin L and serine protease activities detected in this species could be related to its greater pathogenicity, reported in experimental animals, compared to that of A. pegreffii.