Abstract
Proteases and their inhibitors involved in the metamorphoses of zoonotic Anisakis pegreffii larvae and infection success of paratenic and accidental hosts
Anisakiasis is a fish-borne zoonosis caused by the accidental ingestion of live and/or dead third-stage larvae (L3) of the genus Anisakis Dujardin, 1845, and it is considered fifth in the European risk ranking and the second of 24 foodborne parasitoses with the highest “increasing illness potential”. Although human is not its final and targeted host, severe gastrointestinal symptoms, abdominal pain or allergic manifestations may arise as a result of the nematode’s attempt to migrate through human digestive mucosa. In parasitic Nematoda, excretory gland cell is tightly related to the pathogenicity of the species by producing a multitude of bioactive excretory and secretory products enabling larval penetration, migration, and potentially feeding. De novo assembly of A. pegreffii genome, complemented with the transcriptomics of the parasite major excretory and secretory gland profiled through parasite ontogeny and invasion of paratenic and accidental host models (seabass and rat), enabled us to prospect nematode’s major virulence factors within peptidases and peptidase inhibitors (PPI) families. Combining for the first time the long-read PromethION and short-read Illumina sequencing approach, we propose a draft genome of A. pegreffii of 128.84 Mb, 3,070 contings, with a contig N50 of 1,953 bp and GC content of 44.11%. The annotation includes 13,914 protein-coding genes and 1,078 non-coding RNA genes. Based on functional annotation, GO terms, presence of specific domains and BLAST searchers with MEROPS and UNIREF100 database, a strategy was developed to scrutinize peptidases and peptidase inhibitors. During moulting of L3 to L4, the expression of 262 proteases and 28 protease inhibitors was registered in the excretory gland cell, with metalloproteases and serin proteases as predominant types, of which 103 were differentially regulated. Most strongly upregulated were prolylcarboxypeptidases, while homologues of astacins, different metallopeptidases and autophagin were downregulated. In accordance with general transcriptional activity observed during infection of accidental poikilothermic host (rat) and paratenic (seabass), over a hundred peptidases and inhibitors were regulated during L3 attempt to infect a rat, and much less (17) in seabass. This confirms that important virulence factors in A. pegreffii are closely connected and play a role in its development and maturation and sets a ground for better understanding of its pathogenesis.