Transcriptomic analysis supports a role for the nervous system in regulating growth and development of Fasciola hepatica juveniles

Abstract

Fasciola spp. liver flukes have significant impacts in veterinary and human medicine. The absence of a vaccine and increasing anthelmintic resistance threaten sustainable control and underscore the need for novel flukicides. Functional genomic approaches underpinned by in vitro culture of juvenile Fasciola hepatica facilitate control target validation in the most pathogenic life stage. Comparative transcriptomics of in vitro and in vivo maintained 21 day old F. hepatica finds that 86% of genes are expressed at similar levels across maintenance treatments suggesting commonality in core biological functioning within these juveniles. Phenotypic comparisons revealed higher cell proliferation and growth rates in the in vivo juveniles compared to their in vitro counterparts. These phenotypic differences were consistent with the upregulation of neoblast-like stem cell and cell-cycle associated genes in in vivo maintained worms. The more rapid growth/development of in vivo juveniles was further evidenced by a switch in cathepsin protease expression profiles, dominated by cathepsin B in in vitro juveniles and by cathepsin L in in vivo juveniles. Coincident with more rapid growth/development was the marked downregulation of both classical and peptidergic neuronal signalling components in in vivo maintained juveniles, supporting a role for the nervous system in regulating liver fluke growth and development. Differences in the miRNA complements of in vivo and in vitro juveniles identified 31 differentially expressed miRNAs, including fhe-let-7a-5p, fhe-mir-124-3p and miRNAs predicted to target Wnt-signalling, which supports a key role for miRNAs in driving the growth/developmental differences in the in vitro and in vivo maintained juvenile liver fluke. Widespread differences in the expression of neuronal genes in juvenile fluke grown in vitro and in vivo expose significant interplay between neuronal signalling and the rate of growth/development, encouraging consideration of neuronal targets in efforts to dysregulate growth/development for parasite control.

Fasciola hepatica juveniles interact with the host fibrinolytic system as a potential early-stage invasion mechanism

“…Notwithstanding the origin of the tegument PLG-binding proteins, and based on the capacity of plasmin to degrade components of the ECM [35], these results support our original idea that PLG fixation on the tegument could represent a mechanism for FhNEJ to harness the functions of the host fibrinolytic system that provides an additional source of proteolytic activity to facilitate traversal of the intestinal wall and the successful establishment of the parasite within the vertebrate host. In the future, these findings could be further validated using in vivo or ex vivo models of F. hepatica infection [36,37].…”

Discovery of long non-coding RNAs in the liver fluke,Fasciola hepatica

“…DE transcripts were found in all pairwise comparisons, with the number of DE transcripts roughly proportional to the time-course length between stages -the fewest between met:nej1h (5 lncRNA, 145 mRNA), and the most between juv21d:met (822 lncRNA, 2104 mRNA). Comparison of juv21d with invitro21d samples (respectively, flukes recovered from rat livers 21 days after infection, or maintained in vitro for 21 days (McCusker et al, 2016;Robb et al, 2022), demonstrated the presence of 13 lncRNAs found uniquely in invitro21d samples, with 63 found uniquely in juv21d worms. Given the key developmental differences between these groups (Robb et al, 2022), these lncRNAs have potential importance for fluke development, and represent priority targets for functional genomics experiments.…”

“…Comparison of juv21d with invitro21d samples (respectively, flukes recovered from rat livers 21 days after infection, or maintained in vitro for 21 days (McCusker et al, 2016; Robb et al, 2022), demonstrated the presence of 13 lncRNAs found uniquely in invitro21d samples, with 63 found uniquely in juv21d worms. Given the key developmental differences between these groups (Robb et al, 2022), these lncRNAs have potential importance for fluke development, and represent priority targets for functional genomics experiments.…”

“…We supplemented these with stranded paired-end Illumina sequencing datasets for rat-derived juv21d worms (juv21d; n=3 ), and 21 day in vitro maintained worms (invitro21d; n =3). These six libraries are available from ENA project PRJEB49655, samples ERS9656160-ERS9656165, generated as described in (Robb et al, 2022). The F. gigantica 18h NEJ RNA-Seq dataset was generated as described in (Davey et al, 2022), available from DDBJ/EMBL/GenBank accession GJHP01000000 and can be interrogated via https://sequenceserver.ibers.aber.ac.uk/.…”

“…This produced 120,927 transcripts of ≧ 200 nt length (the default length cutoff within StringTie), which were passed into our lncRNA annotation pipeline (Figure 1). A separate stranded transcriptome was also produced by mapping the three (Robb et al, 2022) juv21d datasets against the F. hepatica genome as described above, and employing the - -fr option in StringTie to denote a stranded library in “fr-firststrand” format, matching the dUTP-based synthesis method of the stranded library synthesis kit that we used (Robb et al, 2022).…”