Biotinylation of the Neospora caninum parasitophorous vacuole reveals novel dense granule proteins

Congshan Yang,Chenrong Wang,Qun Liu,Jing Liu

doi:10.1186/s13071-021-05023-7

Abstract

BackgroundNeospora caninum is an obligate intracellular parasite that invades host cells and replicates within the parasitophorous vacuole (PV), which resists fusion with host cell lysosomal compartments. To modify the PV, the parasite secretes an array of proteins, including dense granule proteins (GRAs). The vital role of GRAs in the Neospora life cycle cannot be overestimated. Despite this important role, only a subset of these proteins have been identified, and most of their functions have not been elucidated. Our previous study demonstrated that NcGRA17 is specifically targeted to the delimiting membrane of the parasitophorous vacuole membrane (PVM). In this study, we utilize proximity-dependent biotin identification (BioID) to identify novel components of the dense granules.MethodsNcGRA17 was BirA* epitope-tagged in the Nc1 strain utilizing the CRISPR/Cas9 system to create a fusion of NcGRA17 with the biotin ligase BirA*. The biotinylated proteins were affinity-purified for mass spectrometric analysis, and the candidate GRA proteins from BioID data set were identified by gene tagging. To verify the biological role of novel identified GRA proteins, we constructed the NcGRA23 and NcGRA11 (a–e) knockout strains using the CRISPR/Cas9 system and analyzed the phenotypes of these mutants.ResultsUsing NcGRA17-BirA* fusion protein as bait, we have identified some known GRAs and verified localization of 11 novel GRA proteins by gene endogenous tagging or overexpression in the Nc1 strain. We proceeded to functionally characterize NcGRA23 and NcGRA11 (a–e) by gene knockout. The lack of NcGRA23 or NcGRA11 (a–e) did not affect the parasite propagation in vitro and virulence in vivo.ConclusionsIn summary, our findings reveal that BioID is effective in discovering novel constituents of N. caninum dense granules. The exact biological functions of the novel GRA proteins are yet unknown, but this could be explored in future studies.Graphical abstract

Highlights

Neospora caninum is an obligate intracellular parasite that invades host cells and replicates within the parasitophorous vacuole (PV), which resists fusion with host cell lysosomal compartments
NcGRA17 was BirA* epitope-tagged in the Nc1 strain utilizing the CRISPR/ Cas9-directed genome editing system to create a fusion of NcGRA17 with the biotin ligase BirA* (Fig. 1a; Additional file 1: Figure S1a–c)
We observed staining in the PV (Fig. 1b), a pattern consistent with NcGRA7, indicating that the fusion did not alter the trafficking of NcGRA17

Summary

Introduction

Neospora caninum is an obligate intracellular parasite that invades host cells and replicates within the parasitophorous vacuole (PV), which resists fusion with host cell lysosomal compartments. The vital role of GRAs in the Neospora life cycle cannot be overestimated Despite this important role, only a subset of these proteins have been identified, and most of their functions have not been elucidated. Our previous study demonstrated that NcGRA17 is targeted to the delimiting membrane of the parasitophorous vacuole membrane (PVM). There are three localizations in PVs within the infected cell where Neospora dense granule proteins (NcGRAs) are exhibited: the vacuolar space, intravacuolar membranous nanotubular network (MNN), and parasitophorous vacuole membrane (PVM). We adapted the proximity-dependent biotin identification (BioID) system for Neospora and used the dense granule protein NcGRA17 as bait to identify novel NcGRA proteins. The discovery of novel NcGRA proteins provides a more comprehensive basis for the understanding of host–parasite interactions and N. caninum parasitism

Methods

Results

Discussion

Conclusion