Transcriptomic and proteomic profiling of peptidase expression in Fasciola hepatica eggs developing at host’s body temperature

Abstract

Fasciola hepatica is a global parasite of livestock which also causes a neglected zoonosis in humans. The parasite’s communication with the host during its complicated lifecycle is based on an ingenious enzymatic apparatus which includes a variety of peptidases. These enzymes are implicated in parasite migration, pathogenesis of the disease, and modification of host immune response. Although the dynamics of proteolytic machinery produced by intra-mammalian F. hepatica life stages has been previously investigated in great detail, peptidases of the eggs so far received little scientific attention. In this study, we performed a comparative RNA-seq analysis aimed at identification of peptidases expressed in F. hepatica eggs, cultured at 37 °C to represent gall bladder retained eggs, for different time periods and employed mass spectrometry in order to identify and quantify peptidases translated in F. hepatica egg lysates. We demonstrated that F. hepatica eggs undergo significant molecular changes when cultured at the physiological temperature of the definitive host. Egg transcriptome is subject to numerous subtle changes while their proteome is even more variable. The peptidase profile is considerably modified on both transcriptome and proteome level. Finally, we measured and classified proteolytic activities in extracts from F. hepatica eggs using a library of fluorogenic substrates and peptidase class-selective inhibitors. Activities of threonine peptidases were detected constantly, while the cysteine peptidases prevailing in freshly laid eggs are substituted by aspartic peptidase and metallopeptidase activities in the later stages of egg development.

Exploiting Comparative Omics to Understand the Pathogenic and Virulence-Associated Protease: Anti-Protease Relationships in the Zoonotic Parasites Fasciola hepatica and Fasciola gigantica

“…In agreement with the study by Ilgová et al [ 30 ], we observed that the eggs express a more dynamic range of peptidases and peptidase inhibitors, and this is also exhibited by the miracidia and rediae. This analysis is also consistent with the transcriptome analysis by Zhang et al [ 26 ] that described an upregulation of peptidase inhibitors in the egg transcriptome, and an enrichment of zinc ion binding and metallo-endopeptidase activity.…”

“…Despite observing lower transcriptional levels of metallopeptidase inhibitors (I63) in F. hepatica eggs compared to F. gigantica , protein products of these genes are abundant within the F. hepatica egg somatic proteome. It is worthwhile noting, however, as highlighted in the study by Ilgová et al [ 30 ], that Fasciola eggs are laid unembryonated and as the eggs develop, their transcriptional and protein profiles change.…”

“…In this study we have re-analysed our previously published F. hepatica and F. gigantica omics datasets to identify the peptidase and peptidase inhibitor genes [ 12 , 26 , 27 , 28 , 29 ] and carried out comparative analyses with the recently available F. hepatica egg transcriptome and proteome data published by Ilgová et al [ 30 ] and F. gigantica genome assemblies [ 14 , 15 ] as detailed below.…”

“…Analysis by BLASTp against the MEROPS collection (release 11.0; , accessed on 30 August 2022; default settings, accessed 21 July 2022 [ 31 ]) followed by manual annotation was carried out to determine the peptidase and peptidase inhibitor gene family profile using the following Fasciola spp. datasets: (1) F. hepatica analysis was based on the gene models identified within the F. hepatica genome (PRJEB6687; PRJEB25283; [ 12 ]) and by parsing the data from the study of the F. hepatica egg transcriptome (GSE160622) published by Ilgová et al [ 30 ]; (2) F. gigantica stage-specific transcriptomes (PRJNA350370) reported by Zhang et al [ 26 ]. The transcript and protein expression profiles were extracted from the stage-specific transcriptome and proteome datasets for F. hepatica [ 12 , 27 , 28 , 29 ] and F. gigantica [ 26 ].…”

“…life cycle. ( A , B ) The transcriptional profile of F. hepatica based on the percentage transcripts per million (TPM) of the respective peptidase ( A ) and peptidase inhibitors families ( B ) from the stage-specific transcriptomes described by [ 12 , 30 ]. ( C , D ) The transcriptional profile of F. gigantica based on the percentage fragments per kilobase of exon per million mapped fragments (FPKM) of the respective peptidase ( C ) and peptidase inhibitors families ( D ) from the stage-specific transcriptomes described by [ 26 ].…”