Author:
Yang Congshan,Wang Chenrong,Liu Jing,Liu Qun
Abstract
Abstract
Background
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. 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.
Methods
NcGRA17 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.
Results
Using 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.
Conclusions
In 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
Funder
National Key Research and Development Program of China
National Natural Science Foundation of China
Natural Science Foundation of Anhui Province
Publisher
Springer Science and Business Media LLC
Subject
Infectious Diseases,Parasitology
Reference30 articles.
1. Dubey JP, Carpenter JL, Speer CA, Topper MJ, Uggla A. Newly recognized fatal protozoan disease of dogs. J Am Vet Med Assoc. 1988;192:1269–85.
2. Lefkaditis M, Mpairamoglou R, Sossidou A, Spanoudis K, Tsakiroglou M. Neospora caninum, a potential cause of reproductive failure in dairy cows from northern greece. Vet Parasitol Reg Stud Rep. 2020;19: 100365.
3. Reichel MP, Ayanegui-Alcérreca MA, Gondim LFP, Ellis JT. What is the global economic impact of Neospora caninum in cattle—the billion dollar question. Int J Parasitol. 2012;43:133–42.
4. Dubey JP, Schares G, Ortega-Mora LM. Epidemiology and control of neosporosis and Neospora caninum. Clin Microbiol Rev. 2007;20:323–67.
5. Cesbron-Delauw MF. Dense-granule organelles of Toxoplasma gondii: their role in the host–parasite relationship. Parasitol Today. 1994;10:293–6.
Cited by
6 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献