Targeted proteomics and specific immunoassays reveal the presence of shared allergens between the zoonotic nematodes Anisakis simplex and Pseudoterranova decipiens

Abstract

The family Anisakidae, mainly represented by Anisakis simplex s.l. and Pseudoterranova decipiens, encompasses zoonotic nematodes infecting many marine fish. Both are responsible for gastrointestinal disease in humans after ingestion of a live larva by consumption of undercooked fish, and, in the case of A. simplex, an allergic reaction may occur after consuming or even handling infected fish. Due to its phylogenetic relatedness with A. simplex, few studies investigated the allergenic potential of P. decipiens, yet none of them focused on its excretory/secretory (E/S) proteins that easily get missed when working solely on extracts from crushed nematodes. Moreover, these E/S allergens remain behind even when the larva has been removed during fish quality processing. Therefore, the aim was to investigate if Anisakis-like allergens could also be detected in both crushed and E/S P. decipiens protein extract using targeted mass spectrometry analysis and immunological methods. The results confirmed that at least five A. simplex allergens have homologous proteins in P. decipiens; a result that emphasizes the importance of also including E/S protein extracts in proteomic studies. Not only A. simplex, but also P. decipiens should therefore be considered a potential source of allergens that could lead to hypersensitivity reactions in humans.

Figure 1

Transition peaks on Skyline for precursor peptides from Ani s 2.0101, Ani s 5.0101, Ani s 7.0101, Ani s 8.0101, and Ani s 13.0101 detected in Pseudoterranova decipiens excretory/secretory and crude worm protein extracts (ESP and CrP, respectively). Each color represents a different transition (i.e., precursor peptide/fragment ion pair).

Figure 2

Alignment of the Ani s 7.0101 precursor peptide YGIEFCNR detected in the Pseudoterranova decipiens s.s. excretory-secretory protein extract (ESP) with the amino acid sequence from the protein ABL77410.1 (partial Ani s 7.0101) and the amino acid sequence (P3), both reported to be recognized by mAb UA3.

Figure 3

(A) Western blot analysis of A. simplex (lane 1) and P. decipiens (lane 3) crude protein extract with monoclonal antibody (mAb) UA3, and after inhibition with peptide P3 (lane 2 and 4, respectively). Arrowheads indicate the position of the common major band recognized by the mAb UA3 in both extracts. (B) BlueSafe-stained SDS-PAGE of A. simplex (lane 5) and P. decipiens (lane 7) crude protein extracts remaining proteins after blotting. Lane 6 shows the migration of the molecular weight marker corresponding to the values indicated at the left of the figure. Lanes in the figure were cropped from whole gel/blots images as shown in Figure S2 for a better definition of the high molecular weight region where mAb UA3 reacts with Anisakis antigens.