Proteomic characterization of adult Fasciola gigantica recovered from naturally infected buffaloes in Punjab Pakistan.

Abstract

Fasciola gigantica (F. gigantica) is a widespread zoonotic trematode that poses a substantial burden on livestock and public health in endemic regions. Elucidating host-parasite interactions relies largely on proteomic data. Excretory-Secretory proteins (ESPs) can play central roles in the host-parasite interactions, immune evasion, and parasite pathogenicity; however, proteomic information on F. gigantica infecting buffalo remains scarce, particularly from Pakistan. In this study, we present the first comprehensive proteomic characterization of adult F. gigantica recovered from naturally infected buffaloes in Punjab, Pakistan, with a specific focus on putative excretory-secretory proteins. Somatic protein extracts from adult flukes were initially assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting, confirming a high abundance and broad molecular weight distribution of parasite proteins. Subsequent liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis generated the largest proteomic dataset for F. gigantica to date, a total of 4,777 proteins. In‒silico prediction using DeepLoc 2.0, SignalP, and transmembrane helices (TMHMM), enabled the identification of a substantial repertoire of putative ESPs, including both classical secretory proteins possessing N-terminal signal peptides and non-classical secreted proteins lacking transmembrane helices. Functional annotation and Gene Ontology (GO) enrichment analysis revealed that these proteins were predominantly associated with catalytic, hydrolase, and oxidoreductase activities, highlighting their roles in tissue invasion and nutrient acquisition. Among 4,777 proteins, 95 proteins associated with molecular functions, 77 in biological processes, and 65 in cellular components were linked to immune regulation, stress response, metabolic adaptation, and parasite survival. Pathway enrichment further highlighted their involvement in protein processing, signal transduction, and mechanisms facilitating host invasion and chronic infection. Collectively, these findings emphasize the vital role of proteins (somatic/putative secreted) in orchestrating host-parasite interactions and sustaining chronic infection.