Know your enemy - transcriptome of myxozoan Tetracapsuloides bryosalmonae reveals potential drug targets against proliferative kidney disease in salmonids

Abstract

The myxozoan Tetracapsuloides bryosalmonae is a widely spread endoparasite that causes proliferative kidney disease (PKD) in salmonid fish. We developed an in silico pipeline to separate transcripts of T. bryosalmonae from the kidney tissue of its natural vertebrate host, brown trout (Salmo trutta). After stringent filtering, we constructed a partial transcriptome assembly T. bryosalmonae, comprising 3427 transcripts. Based on homology-restricted searches of the assembled parasite transcriptome and Atlantic salmon (Salmo salar) proteome, we identified four protein targets (Endoglycoceramidase, Legumain-like protease, Carbonic anhydrase 2, Pancreatic lipase-related protein 2) for the development of anti-parasitic drugs against T. bryosalmonae. Earlier work of these proteins on parasitic protists and helminths suggests that the identified anti-parasitic drug targets represent promising chemotherapeutic candidates also against T. bryosalmonae, and strengthen the view that the known inhibitors can be effective in evolutionarily distant organisms. In addition, we identified differentially expressed T. bryosalmonae genes between moderately and severely infected fish, indicating an increased abundance of T. bryosalmonae sporogonic stages in fish with low parasite load. In conclusion, this study paves the way for future genomic research in T. bryosalmonae and represents an important step towards the development of effective drugs against PKD.

Keywords: Aquatic pathogens; Myxozoa; climate change; dual RNA-seq; minicollagen; next-generation sequencing; salmonid fish.

Graphical abstract

Fig. 1.

Summary of the taxonomic classification of assembled transcripts. Bar plots in (A) indicate the number of transcripts assigned (or unclassified) to the most frequent GenBank's taxonomic divisions. Venn diagrams illustrate the classification agreement between the BLASTN, BLASTX and Kraken based assignments. Within the invertebrates and vertebrates, only myxozoans and bony fish are shown in the Venn diagrams, respectively. The bar plots in (B–D) show the species to which most of the transcripts were assigned by BLASTN, BLASTX and Kraken, respectively.

Fig. 2.

Characterization of T. bryosalmonae transcripts: (A) The distributions of GC content of all assembled transcripts (grey; n = 14741; mean GC = 35.51%), putative parasitic sequences (light grey; n = 3427; mean GC = 31.67%) and transcripts belonging to bony fishes in BLAST results (black; n = 1027; mean GC = 48.56%). (B) The distribution of Pearson's correlation coefficients between the raw read counts of putative parasite transcripts and the qPCR-inferred relative parasite load.

Fig. 3.

BUSCO single-copy metazoan genes and proteases in Myxozoa (A) Venn diagram showing the number of BUSCO single-copy genes identified (complete + fragmented) in each species. The T and G after the name of species represent de novo assembled transcriptome and CDS extracted from the genomes, respectively. (B) Venn diagram showing the number of BUSCO single-copy genes missing in each species. (C) Distribution of proteases in current T. bryosalmonae transcriptome in relation to earlier studies. The numbers in each category represent protease classes found in each study.

Fig. 4.

Differential transcript expression and GO enrichment: (A) Heatmap showing variance stabilized expression of the differentially expressed transcripts between the moderately and severely infected hosts. (B) Volcano plot showing the fold-change differences between the moderately and severely infected hosts. Light blue and red points represent the down- and up-regulated genes in severely infected fish, respectively.

Fig. 5.

Prediction of the EGCase2 (DN1345_c0_g1_i1) and CA2 (DN419_c0_g2_i1) three-dimensional structures in T. bryosalmonae. (pink). (A) The parasite EGCase2 (green) structure is superimposed on the chain B of the template endoglycoceramidase ii from rhodococcus sp. In EGCase2, the inhibitor (yellow) is shown in ball and stick representation. Similar binding cavities between template and target can be seen around the inhibitor molecule. (B) The same structure shown in ribbons to illustrate the consistency between the template and target structures. (C) The interactions between the EGCase II amino acids and inhibitor at the binding site are shown with black lines as reported in Caines et al. (2007). (D) The parasite CA2 (green) structure is superimposed on the chain A of the Schistosoma mansoni carbonic anhydrase (SmCA, pink). The surface is made transparent to show the active site. (E) The same structure shown in ribbons to illustrate the consistency between SmCA and the predicted T. bryosalmonae structures. (F) The active site (His⁷⁶, Gln¹⁰⁷, Glu¹²², and Thr missing) and Zinc binding Histidines (His¹⁰⁹, His¹¹¹ and His¹¹³⁶) residues of T. bryosalmonae CA2 also acquired similar orientation as the SmCA (Da'dara et al., 2019).