The pervasive effects of recombinant Fasciola gigantica Ras-related protein Rab10 on the functions of goat peripheral blood mononuclear cells

Abstract

Background: Fasciola gigantica-induced immunomodulation is a major hurdle faced by the host for controlling infection. Here, we elucidated the role of F. gigantica Ras-related protein Rab10 (FgRab10) in the modulation of key functions of peripheral blood mononuclear cells (PBMCs) of goats.

Methods: We cloned and expressed recombinant FgRab10 (rFgRab10) protein and examined its effects on several functions of goat PBMCs. Protein interactors of rFgRab10 were predicted in silico by querying the databases Intact, String, BioPlex and BioGrid. In addition, a total energy analysis of each of the identified interactions was also conducted. Gene Ontology (GO) enrichment analysis was carried out using FuncAssociate 3.0.

Results: The FgRab10 gene (618 bp), encodes 205-amino-acid residues with a molecular mass of ~23 kDa, had complete nucleotide sequence homology with F. hepatica Ras family protein gene (PIS87503.1). The rFgRab10 protein specifically cross-reacted with anti-Fasciola antibodies as shown by Western blot and immunofluorescence analysis. This protein exhibited multiple effects on goat PBMCs, including increased production of cytokines [interleukin-2 (IL-2), IL-4, IL-10, transforming growth factor beta (TGF-β) and interferon gamma (IFN-γ)] and total nitric oxide (NO), enhancing apoptosis and migration of PBMCs, and promoting the phagocytic ability of monocytes. However, it significantly inhibited cell proliferation. Homology modelling revealed 63% identity between rFgRab10 and human Rab10 protein (Uniprot ID: P61026). Protein interaction network analysis revealed more stabilizing interactions between Rab proteins geranylgeranyltransferase component A 1 (CHM) and Rab proteins geranylgeranyltransferase component A 2 (CHML) and rFgRab10 protein. Gene Ontology analysis identified RabGTPase mediated signaling as the most represented pathway.

Conclusions: rFgRab10 protein exerts profound influences on various functions of goat PBMCs. This finding may help explain why F. gigantica is capable of provoking recognition by host immune cells, less capable of destroying this successful parasite.

Keywords: Fasciola gigantica; Immunomodulation; Peripheral blood mononuclear cells; Ras-related protein Rab10; Recombinant proteins.

Fig. 1

SDS-PAGE and Western blot analyses of the purified rFgRab10 protein from the sonicated culture sediment of E. coli. a rFgRab10 protein was resolved on 12% acrylamide gel and stained with Coomassie blue. Lane M: prestained protein ladder in kilodaltons; Lane P: purified recombinant protein, rFgRab10, which appeared as a single band of ~39 kDa. b rFgRab10 protein was run under non-reducing conditions, and the immunoreaction was visualized using chemiluminescent HRP substrate and X-ray film. Lane M: prestained protein ladder; Lane P: loaded with rFgRab10 protein expressed from E. coli. Serum from Fasciola-infected sheep detected a single band of ~39 kDa; Lane C1: loaded with protein expressed from sonicated culture supernatant of E. coli transformed with empty pET-SUMO vector without rFgRab10 gene insert, which did not react with goat serum containing anti-F. gigantica IgG antibodies; Lane C2: loaded with rFgRab-10 protein expressed from E. coli that did not react with the serum of healthy uninfected goats

Fig. 2

Localization of F. gigantica-derived rFgRab10 protein to the PBMCs surface. rFgRab10 protein binding to goat PBMCs surface was determined by incubating PBMCs treated with purified rFgRab10 protein, protein from sonicated culture of E. coli transformed with empty pET-SUMO vector or sham-treated with PBS (control) with primary anti-rFgRab-10 protein antibody raised in rats. PBMCs’ nuclei and rFgRab10 protein were stained by Hoechst 33342 (blue) and Cy3-conjugated secondary antibody (red), respectively. Surface staining was detected in rFgRab10-treated cells, but not in PBMCs incubated with SUMO protein and sham-treated PBMCs. Scale-bars: 10 μm

Fig. 3

rFgRab10 protein induced the cytokine secretion. Goat PBMCs were incubated for 24 h in the presence of serial concentrations of rFgRab10 protein. PBMCs incubated with SUMO protein or PBS-treated PBMCs served as controls. The levels of cytokine concentration in the supernatant of cultured PBMCs was quantified by ELISA. Graphs represent means ± SD of data from three independent biological replicates. Asterisks indicate statistical significance between the different indicated groups (**P < 0.01; ****P < 0.0001; ns, non-significant)

Fig. 4

Effects of rFgRab10 protein on the production of intracellular NO. PBMCs were sham-treated with PBS, SUMO protein or serial concentrations of rFgRab10 protein and incubated for 24 h at 37 °C at 5% CO₂. NO concentration in the PBMCs was measured using Griess assay. Graphs represent means ± SD of data from 3 independent biological replicates. Asterisks indicate statistical significance between the different indicated groups (****P < 0.0001; ns, non-significant)

Fig. 5

rFgRab10 protein induced apoptosis in PBMCs. Annexin V/PI staining combined with flow cytometry analysis was used to count apoptotic cells. a Dot plots showing apoptosis of PBMCs in response to exposure to rFgRab10 protein. b Percentage of cell population was compared with plotted apoptotic cells (Annexin V+/PI-). Graphs represent means ± SD of data from three independent biological replicates. Asterisks indicate statistical significance between rFgRab10-treated, SUMO protein and sham-treated PBMCs (****P < 0.0001; ns, non-significant). The statistical significance was only achieved at the two highest concentrations (40 μg/ml and 80 μg/ml)

Fig. 6

rFgRab10 protein promoted goat PBMCs migration. PBMCs were treated with PBS, SUMO protein or serial concentrations of rFgRab10 protein and then the goat PBMCs migration percentage (%) was determined. Graphs represent means ± SD of data from 3 independent biological replicates. Asterisks indicate statistical significance between the different indicated groups (*P < 0.05; **P < 0.01; ****P < 0.0001; ns, non-significant)

Fig. 7

rFgRab10 protein promoted phagocytosis of goat monocytes. rFgRab10 protein increased the phagocytic ability of goat monocytes as indicated by the increase in FITC-dextran uptake in goat monocytes treated with serial concentrations of rFgRab10 protein, compared with those treated with PBS or SUMO protein. Graphs represent means ± SD of data from three independent biological replicates. Asterisks indicate statistical significance between the different indicated groups (***P < 0.001; ****P < 0.0001; ns, non-significant)

Fig. 8

rFgRab10 protein inhibited goat PBMCs proliferation. Goat PBMCs were treated with PBS, SUMO protein or serial concentrations of rFgRab10 protein and incubated for 48 h at 37 °C at 5% CO₂. Proliferation of goat PBMCs was determined using CCK-8 assay. Results indicated that rFgRab10 protein significantly inhibited PBMCs proliferation. Mean values and the respective standard deviation of three independent experiments are presented. Asterisks indicate statistical significance between the different indicated groups (****P < 0.0001; ns, non-significant)

Fig. 9

De novo 3D ab initio model for rFgRab10 protein. The model was generated using Rosetta

Fig. 10

The structure alignment of rFgRab10 to the human Rab10 protein. The predicted rFgRab10 model shared 63% similarity with the human Rab10 protein (Uniprot ID: P61026)

Fig. 11

3D representation of both rFgRab10 protein and human Rab10 protein: Chain A of human Rab10 protein (red) and rFgRab10 protein (blue), with the aligned region shown in yellow

Fig. 12

The protein interaction network revealed more stable interactions, compared to the human ortholog Rab10, between Rab proteins geranylgeranyltransferase component A 1 (CHM) and Rab proteins geranylgeranyltransferase component A 2 (CHML) and rFgRab10 sequence (red edges); whereas fewer stable interactions were detected between [F-actin]-monooxygenase MICAL3 (MICAL3) and Guanine nucleotide exchange factor MSS4 (RABIF) proteins and rFgRab10 (blue edges)