Authors

J W Holland⁴; S Yoon⁴; M Faber⁴; H Hartikainen³; B Abos¹; I Estensoro²; C Bailey⁵; K A Veenstra⁴; A Alnabulsi⁴; T Wang⁴; C Tafalla¹; C J Secombes⁴;  ¹ Centro de Investigación en Sanidad Animal, Spain;  ² Institute of Aquaculture Torre de la Sal, Spain;  ³ Institute of Aquatic Ecology, Switzerland;  ⁴ University of Aberdeen, UK;  ⁵ University of Bern, Switzerland

Discussion

The Myxozoa represents a large group of enigmatic endoparasites sharing morphological features with bilateria, protists, and cnidarians. Due to their extreme sequence diversity, precise phylogenetic placement has only recently been resolved placing them firmly in the phylum, cnidaria. Our work has focused on Tetracapsuloides bryosalmonae, a myxozoan that cycles between an invertebrate host (colonial bryozoans) and salmonid fish causing proliferative kidney disease (PKD) in the latter. The chronic pathology associated with PKD is refractory towards pro-inflammatory mechanisms, although eliciting dominant anti-inflammatory activities, and dysregulated B and T cell responses. Our recent investigations have focused on putative T. bryosalmonae virulence factors to attempt to unravel host immune evasion mechanisms exploited by the parasite.

A unique form of antigenic diversification that differs to classical antigenic variation, has been described in schistosomes based on the variant expression of proteins encoded by micro-exon genes (MEGs). MEGs are stage-specific intrinsically disordered secreted proteins associated with external parasite surfaces that are likely to be in contact with the host immune system. Unlike other micro-exon containing genes, > 75% of the coding region of MEGS consists of very short exons that undergo alternative splicing to yield an array of protein variants. It has been postulated that protein variants may have numerous host-derived binding partners and so may represent an important form of immune evasion. Indeed, recently a MEG has been shown to potently suppress pro-inflammatory mechanisms via interaction with the human calcium binding protein, S100A9.

Here we report the sequencing and characterisation of the first non-helminth MEG (Tb-MEG1). The 264 amino acid open reading frame of the full length molecule is encoded by 65 exons, with only 5 exons > 30 bp. The predicted structure of Tb-MEG1 is consistent with schistosome MEGs in possessing a signal peptide, a mature protein largely consisting of an intrinsically disordered loop, and an alpha helix tail at the C terminal encoded by the largest exon (145 bp). The intrinsic loop is characterised by the presence of a tandem repeat region. Predominantly expressed in the fish host at both transcriptional and protein levels, we have uncovered numerous cDNA variants expressed in infected fish based on alternative splicing in the repeat region. Consistent with schistosome MEGs and other surface proteins involved in protein-protein interactions, the repeat region possesses 10 putative O-linked glycosylation sites and 4 sulphated tyrosines. By immunohistochemistry we have demonstrated the protein to be endogenously expressed in and on the surface of parasites within the kidneys of infected fish. Intriguingly, the protein has also been found to envelope clusters of lymphoid-like cells, a phenomenon also observed in kidney cells incubated with recombinant Tb-MEG1. We have also demonstrated a potent Tb-MEG1-specific antibody response in parasite-infected farmed rainbow trout that has been corroborated using a lab-based challenge model.

Current studies are in progress to establish host binding partners of Tb-MEG1, the modulation of host immune gene expression by recombinant Tb-MEG1, and its immune protective efficacy as both DNA and protein-based vaccines. Overall, the discovery and characterisation of Tb-MEG1 may provide insights into host immune evasion mechanisms exploited by myxozoan parasites whilst having major implications concerning the evolution of antigenic diversity in metazoan parasites.