Nematodes of the genus Anisakis are the causative agents of anisakiasis, an emerging fish-borne zoonotic disease, characterized by gastrointestinal, ectopic or allergic reactions, following the ingestion of infected raw or undercooked fishes. Despite its increasing public health awareness, most of the mechanisms of infection and clinical outcomes in humans are still unknown. Establishment of successful long-term infections by pathogens usually involves manipulation of host immune responses. Exosomal microvesicles (carrying proteins, DNA and non-coding RNAs) recently emerged as relevant players in intercellular signalling and parasite-host interactions. To identify molecules putatively involved in penetration of host tissues, we recently completed Anisakis larval and pharyngeal transcriptome analyses. To obtain a deeper understanding of molecules involved in host manipulation, we propose to characterize the Anisakis exosomal miRNAs content. MiRNAs play crucial roles in post-transcriptional regulation and pathogens also exploit them to target host genes. Within exosomes, miRNAs travel in a protected state and, as reported for the nematode Brugia malayi, they may actively target host responses to infection. So far, nothing is known on Anisakis exosomal miRNAs and their role in host-parasite interaction. Using RNAseq and cutting-edge bioinformatics, we propose here the characterization of miRNAs from Anisakis larvae and their released exosomes. This is expected to provide the first Anisakis miRNA catalogue, allowing comparative analyses with other nematodes and the identification of potentially targeted human genes. The establishment of a reliable exosome purification protocol will also pave the way for proteomic and electron microscopy studies. Finally, our analysis may shed some light on mechanisms of host manipulation by anisakid nematodes and may help the development of early diagnostic biomarkers, as well as allergen-specific, preventive and therapeutic targets.
With the advent of the Next Generation Sequencing technologies, a large amount of information is becoming available for numerous organisms. Parasitic nematodes have a great biological complexity and a great spectrum of interactions with the host, features that have impeded research progresses on anthelmintic discovery and immunological screening test, as well as on control of infections. The exploration of non-model animals and especially pathogens is fundamental to clarify important issues, such as mechanisms that underlie infectivity.
In fact, the description of a wide-scale pathogenic nematode RNA-seq datasets could be relevant to depict general and specific mechanisms related to pathogenic behaviour, and in a broad-spectrum may favour the development of new diagnostic and screening tools based on antigen-antibody study as well as it may facilitate drugs target discovery. At comparative level, information of this kind will aid in understanding of both conserved and divergent aspects of nematode biology in biomedical research. In this perspective, the present project proposes to characterize the exosomal miRNAs of the Anisakis sp. larval stage, the etiological agent of an emerging fish-borne zoonosis known as anisakiasis.
The disease is worldwide distributed, with major impact in countries with a large consumption of raw fish. Because of unspecific symptoms and diagnostic restrictions, incidence and burden of disease are probably underestimated. Moreover, fish consumption is increasing worldwide and raw preparations as sushi/sashimi or marinated anchovies show a rising trend; lastly, Anisakis exposure has been associated to occupational work risk to develop allergic diseases such as asthma and urticaria. The available and routinely used diagnostic methods for the detection of allergy due to Anisakis (e.g. skin prick test) demonstrated to be poorly specific, as somatic antigens largely share epitopes with other organisms, such as arthropods. The discovery and characterization on highly specific secreted molecules will provide the basis for the improvement of highly specific diagnostic tools for this parasite-driven allergy.
Although the risk related to Anisakis infections is of raising concern, the knowledge on the specific mechanisms underlying host-parasite interactions and on the molecules directly involved in such interactions are still very scarce and the achievement obtained in the present project will represent a repertoire of information of great utility for all researchers involved in such scientific issues.