A recombinant Fasciola gigantica 14-3-3 epsilon protein (rFg14-3-3e) modulates various functions of goat peripheral blood mononuclear cells

Abstract

BackgroundThe molecular structure of Fasciola gigantica 14-3-3 protein has been characterized. However, the involvement of this protein in parasite pathogenesis remains elusive and its effect on the functions of innate immune cells is unknown. We report on the cloning and expression of a recombinant F. gigantica 14-3-3 epsilon protein (rFg14-3-3e), and testing its effects on specific functions of goat peripheral blood mononuclear cells (PBMCs).MethodsrFg14-3-3e protein was expressed in Pichia pastoris. Western blot and immunofluorescence assay (IFA) were used to examine the reactivity of rFg14-3-3e protein to anti-F. gigantica and anti-rFg14-3-3e antibodies, respectively. Various assays were used to investigate the stimulatory effects of the purified rFg14-3-3e protein on specific functions of goat PBMCs, including cytokine secretion, proliferation, migration, nitric oxide (NO) production, phagocytosis, and apoptotic capabilities. Potential protein interactors of rFg14-3-3e were identified by querying the databases Intact, String, BioPlex and BioGrid. A Total Energy analysis of each of the identified interaction was performed. Gene Ontology (GO) enrichment analysis was conducted using Funcassociate 3.0.ResultsSequence analysis revealed that rFg14-3-3e protein had 100% identity to 14-3-3 protein from Fasciola hepatica. Western blot analysis showed that rFg14-3-3e protein is recognized by sera from goats experimentally infected with F. gigantica and immunofluorescence staining using rat anti-rFg14-3-3e antibodies demonstrated the specific binding of rFg14-3-3e protein to the surface of goat PBMCs. rFg14-3-3e protein stimulated goat PBMCs to produce interleukin-10 (IL-10) and transforming growth factor beta (TGF-β), corresponding with low levels of IL-4 and interferon gamma (IFN-γ). Also, this recombinant protein promoted the release of NO and cell apoptosis, and inhibited the proliferation and migration of goat PBMCs and suppressed monocyte phagocytosis. Homology modelling revealed 65% identity between rFg14-3-3e and human 14-3-3 protein YWHAE. GO enrichment analysis of the interacting proteins identified terms related to apoptosis, protein binding, locomotion, hippo signalling and leukocyte and lymphocyte differentiation, supporting the experimental findings.ConclusionsOur data suggest that rFg14-3-3e protein can influence various cellular and immunological functions of goat PBMCs in vitro and may be involved in mediating F. gigantica pathogenesis. Because of its involvement in F. gigantica recognition by innate immune cells, rFg14-3-3e protein may have applications for development of diagnostics and therapeutic interventions.Electronic supplementary materialThe online version of this article (10.1186/s13071-018-2745-4) contains supplementary material, which is available to authorized users.

Modulation of the Functions of Goat Peripheral Blood Mononuclear Cells by Fasciola gigantica Thioredoxin Peroxidase In Vitro

“…F. gigantica discharges many ES molecules that seem to play roles in the induction of this type of immune response. F. gigantica -derived ESPs, identified in the present study (rFgTPx) and in previous studies (rFg14-3-3e [ 20 ] and rFgRab10 [ 21 ]), have induced Th1 (IFN-γ, IL-2) and Th2 (IL-4, IL-10, TGF-β) cytokines, except IL-4 and IFN-γ, whose levels were low in response to rFg14-3-3e. Cytokines are produced by a variety of immune cells and play various roles in protecting the host and maintaining tissue homeostasis [ 28 , 29 ].…”

“…It is readily known that other F. gigantica effector proteins, rFg14-3-3e [ 20 ] and rFgRab10 [ 21 ], alter the phagocytic function of monocytes by decreasing or increasing their phagocytic capacities, respectively, or simply kill PBMCs by inducing apoptosis. By reducing monocyte phagocytosis, rFgTPx would augment the action of rFg14-3-3e [ 20 ] and antagonize the action of rFgRab10 [ 21 ] with regard to the phagocytic capacity of monocytes. By promoting apoptosis at 10–40 μg/mL concentrations, rFgTPx seems to potentiate the action of both rFg14-3-3e and rFgRab10 proteins.…”

“…Adult F. gigantica flukes were obtained from Guangxi Zhuang Autonomous Region (China) as described previously [ 20 ]. Eight local goats (3 to 6 months old) were used in this study.…”

“…Naïve goat sera (used as negative control) were collected from a healthy goat and stored at –80 ℃ for later use. Specific antibodies against rFgTPx were prepared following the protocols described previously [ 20 ], except that rabbits were used in the present study.…”

“…Excretory and secretory products (ESPs), containing many active molecules, have been produced by these parasites under in vitro and in vivo conditions [ 17 ]. Previous reports showed that different molecules of ESPs from F. gigantica (FgESPs) can cause various immune responses—for example, some molecules can simulate host defense against parasites, while other have exerted the opposite effect [ 16 , 18 , 19 , 20 , 21 ]. Of particular importance to their survival is the ability of liver flukes to employ an anti–oxidant, redox-based system that protects the liver flukes against the reactive oxygen and nitrogen species produced by host immune cells and endogenous cellular metabolism [ 22 ].…”