Investigation of blood fluke Infection in bluefin tuna with glycoinformatics
Creators: name: Coff, Lachlan
Publisher: name: RMIT University
Year: 2024
Subjects: classificationCode: 310299
subject: Bioinformatics and computational biology not elsewhere classified
classificationCode: 310407
subject: Host-parasite interactions
classificationCode: 320502
subject: Medical biochemistry - carbohydrates

description: Cardicola forsteri (Digenea: Aporocotylidae) is a blood fluke that parasitises the hearts of bluefin tunas (Thunnus spp.). The prevalence of C. forsteri infection is heightened in mariculture of bluefin tunas, a valuable industry in Japan and Australia. Eggs released from adult C. forsteri become lodged in the gill filaments of the tunas, causing respiratory distress that can result in up to 15% mortality without interventions. Current control measures include moving sea-cages into deeper waters, further away from the sediment-dwelling intermediate hosts (Terebellinae), and treatment with praziquantel (PZQ). As a result of these interventions, mortalities of the tunas have significantly decreased. However, PZQ is not curative, only targeting adults, and does not prevent reinfection. Moreover, the costs incurred from treatment and risk of resistance have prompted the industry to explore alternative control measures, with some sea-cages left untreated. Bluefin tuna have demonstrated the production of TH2-type cytokines and waning cellular immunity in response to infection with Cardicola spp., which is characteristic of modulated host immune responses in other helminthiases, such as schistosomiases. Mechanisms of helminth-induced immunomodulation have not been fully elucidated, but many are thought to involve interactions between parasite glycans and host immune receptors. Other glycoconjugates are targeted by anti-carbohydrate antibodies, and southern bluefin tuna (Thunnus maccoyii) have been shown to mount a non-protective humoral response to unknown antigens in the carbohydrate-rich dorsal tegument of C. forsteri. Little is known about C. forsteri, including the pharmacodynamics of PZQ, so this thesis describes glycomic and genomic investigations of C. forsteri to broaden knowledge that could lead to alternative control measures.

Firstly, a glycoinformatic tool was developed to predict the binding determinants of lectins that were later used in glycoprofiling of C. forsteri. As described in the publication reproduced in Chapter 2, the CCARL (Carbohydrate Classification Accounting for Restricted Linkages) tool assists in mining glycan microarray data for binding motifs of carbohydrate-binding proteins (CBPs), such as lectins. The glycan probes are separated into non-binding, intermediate, and binding sets using the median absolute deviation (MAD) of their relative fluorescence unit (RFU) values, and these class labels are used to train the algorithm. The glycan structures are considered as rooted trees, wherein the monosaccharides are the nodes and the linkages are the edges.

 Additionally, CCARL includes chemically possible linkages that are not present as ‘restricted linkages’. The glycans are then mined for motifs (or subtrees) that are frequent in the binding set relative to the non-binding set in an approach known as ‘frequent subtree mining’. Frequent subtrees are selected using minimum-redundancy, maximum-relevance (mRMR) to return a set of top binding motifs. The inclusion of restricted linkages specifies the location of these motifs within their larger glycan structures, thereby improving their predictive precision over pre-existing frequent subtree mining tools. When tested on glycan microarray data of 20 commercially available lectins, CCARL outperformed the Glycan Miner Tool and GLYMMR with a median area under the curve (AUC) value from receiver operating characteristic (ROC) curves of 0.887. The top binding motifs agreed with binding determinants identified from chromatographic and crystallographic data, even reproducing secondary binding determinants. These results were used to interpret the lectin-reactivity observed for C. forsteri specimens.

The publication reproduced in the Chapter 3.1 describes the first glycomic characterisation of a marine platyhelminth. Mass spectrometry was used to characterise glycans from pooled adult C. forsteri specimens and fluorescent lectin-staining located these glycans at specific organs and tissues. The mass spectrometric analyses revealed a high abundance of oligomannosidic, paucimannosidic, and bi-antennary N-glycans. O-glycans were undetectable, which is consistent with the adult life cycle stage of Schistosoma mansoni. Core fucoses were shown to be α1–6-linked, and lectin-reactivity located these to the tegument of C.forsteri, along with oligomannoses. Additionally, N-glycans carrying terminal N-acetylglucosamine residues allow the hermaphroditic reproductive system to be stained with succinylated wheat germ agglutinin, which could aid morphological characterisations of digeneans. The probability of each lectin used in this study binding to each characterised C. forsteri glycan was determined using CCARL (Chapter 3.2), which validated interpretations of lectin-reactivity. N-glycans carrying core xylose were abundant in adult C. forsteri, whereas these are shed with the cercarial glycocalyx of schistosomes during transformation into schistosomula to avoid immunogenicity in mammalian definitive hosts. The immunogenicity of C. forsteri glycans in bluefin tuna is unknown but could now be investigated with the reactivity of non-naïve bluefin tuna sera to a custom glycan microarray. A novel N-glycan structure carrying a hexuronic acid residue was also characterised, which appears to be the product of an undescribed biosynthetic pathway unique to C. forsteri. The glycosyltransferases involved in the synthesis of these glycans would need to be functionally characterised from an annotated C. forsteri genome assembly.

The manuscript in Chapter 4 describes the sequencing, assembly, and annotation of the whole C. forsteri genome, the first published for an aporocotylid. Prior to this work, genomic information of C. forsteri was limited to three conserved gene sequences used in phylogenetics of the genus. Second- and Third-Generation sequencing technologies were combined to produce a high-quality assembly that bridges the large intergenic repeat regions of the C. forsteri genome. Gene prediction yielded 8,564 protein-coding genes, 1.8% of which were functionally annotated as putative glycosyltransferases and potential drug and vaccine targets. Of most interest were calpains, which have shown efficacy in pre-clinical trials of vaccines against schistosomiasis, and a homologue to a transient receptor potential melastatin (TRPM) that confers sensitivity to PZQ in schistosomes. Annotation of putative fucosyltransferases indicates that, like schistosomes, C. forsteri likely synthesises α1–3-linked fucoses in its pre-adult life cycle stages, including Lewis X and fucosylated LacdiNAc motifs. The C. forsteri genome will be a valuable resource for researchers investigating phylogenetics of the Aporocotylidae and rational drug and vaccine design.

In summary, the glycome of adult C. forsteri was characterised using mass spectrometry and lectin-reactivity, and annotation of its genome revealed glycosyltransferases likely involved in the synthesis of these glycans. Additionally, several potential drug and vaccine candidates were identified, and their immunogenicity in bluefin tuna could be investigated using high-throughput techniques, such as microarrays.