Proteomic Profiling and In Silico Characterization of the Secretome of Anisakis simplex Sensu Stricto L3 Larvae

Abstract

Anisakis simplex sensu stricto (s.s.) L3 larvae are one of the major etiological factors of human anisakiasis, which is one of the most important foodborne parasitic diseases. Nevertheless, to date, Anisakis secretome proteins, with important functions in nematode pathogenicity and host-parasite interactions, have not been extensively explored. Therefore, the aim of this study was to identify and characterize the excretory-secretory (ES) proteins of A. simplex L3 larvae. ES proteins of A. simplex were subjected to liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, and the identified proteins were then analyzed using bioinformatics tools. A total of 158 proteins were detected. Detailed bioinformatic characterization of ES proteins was performed, including Gene Ontology (GO) analysis, identification of enzymes, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways analysis, protein family classification, secretory pathway prediction, and detection of essential proteins. Furthermore, of all detected ES proteins, 1 was identified as an allergen, which was Ani s 4, and 18 were potential allergens, most of which were homologs of nematode and arthropod allergens. Nine potential pathogenicity-related proteins were predicted, which were predominantly homologs of chaperones. In addition, predicted host-parasite interactions between the Anisakis ES proteins and both human and fish proteins were identified. In conclusion, this study represents the first global analysis of Anisakis ES proteins. The findings provide a better understanding of survival and invasion strategies of A. simplex L3 larvae.

Tandem Mass Tagging (TMT) Reveals Tissue-Specific Proteome of L4 Larvae of Anisakis simplex s. s.: Enzymes of Energy and/or Carbohydrate Metabolism as Potential Drug Targets in Anisakiasis

“…Ubiquitin is known to modulate host-pathogen interactions, with a particular focus on host innate immune defenses and pathogen immune evasion [101]. This is consistent with the findings of Kochanowski et al [51], who studied the excretory-secretory proteins of A. simplex larvae. The number of proteins involved in the human-parasite interaction network is about twice the number of proteins in the dolphin-parasite interactome.…”

“…In addition, nine potential allergens were also identified in this study (see Table 2; File S8). One of these, fructose-bisphosphate aldolase (A0A0M3JYW9) was also identified as a potential allergen in the secretome of A. simplex by Kochanowski et al [51]. In addition, many of the potential allergens detected in Anisakis tissues show similarity to the potential allergens identified by other authors in the whole proteome and transcriptome of Anisakis larvae [9,43,45,48,50].…”

“…The number of proteins involved in the human-parasite interaction network is about twice the number of proteins in the dolphin-parasite interactome. This result could be mainly due to the fact that the available database for non-human host-pathogen interactions is much more limited than for human-pathogen interactions, which was also highlighted by Kochanowski et al [51]. Cuticular proteins of A. simplex interacting with the human or dolphin host included serine/threonine protein phosphatase (A0A0M3KIX0; EC 3.1.3.16; STP), 2 peptidase M1 N-domain-containing proteins (A0A3P6NPL4 and A0A3P6PHI6), and the aforementioned iPGM (A0A0M3JK61) (see Figure 7, File S7).…”

“…Therefore, in this study, we described for the first time the proteomic analysis of 2 tissues of the L4 developmental stage of A. simplex s. s. In the L4 stage, tissues, such as the cuticle and intestine, are fully developed and functional [27][28][29]. In addition, previous proteomic studies of A. simplex did not focus on comparative analysis of nematode tissues [41][42][43][44][45][46][47][48][49][50][51]. This work provides new insights into the tissue-specific proteome of A. simplex s. s., which was not previously possible.…”

Inflammatory Response in Caco-2 Cells Stimulated with Anisakis Messengers of Pathogenicity

“…The evidence, here collected about inflammatory pathways after challenges with Anisakis -derived products, revealed interesting and variable outcomes of the cytokines’ production and gene expression. A few hours after ingestion, the L3 release of proteolytic enzymes and chemotactic factors to adhere to and penetrate the mucosa and submucosa [ 47 , 48 ] induced hemorrhagic and erosive lesions [ 7 ] associated with a production of excretory/secretory factors able to interact with the human first line of defense [ 49 ]. The first cells to encounter the invading Anisakis L3 were the epithelial cells, a physical barrier also equipped with microbial-detection mechanisms, signaling the circuits and inflammatory mediators [ 50 ].…”