关键词: 刚地弓形虫, 烯醇酶1, 烯醇酶2, 糖酵解, 速缓殖子转化

Abstract:

Objective To investigate the specific biological functions of Toxoplasma gondii bradyzoite-specific enolase 1 (ENO1) in the tachyzoite-bradyzoite transition, and to analyze the role and molecular mechanism of the ENO family in the regulatory network of T. gondii stage transition. Methods The eno1-deleted strain ME49-Δeno1 and two complementary strains ME49-Δeno1-Comp-eno1 and ME49-Δeno1-Comp-eno2 were generated using the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) gene editing technology. The growth, proliferation, and invasion of four T. gondii strains (ME49-Δku80, ME49-Δeno1, ME49-Δeno1-Comp-eno1, and ME49-Δeno1-Comp-eno2) were evaluated following infections in human foreskin fibroblast cells with the plaque assay, proliferation assay, and invasion assay, respectively. The relative transcriptional levels of tachyzoite-specific surface antigen 1 (sag1) and bradyzoite-specific antigen 1 (bag1) genes were quantified in ME49-Δku80 and ME49-Δeno1 strains using quantitative Real-time PCR (qPCR) assay under in vitro alkaline induction to evaluate the effect of eno1 deletion on the ability of T. gondii tachyzoites transition to bradyzoites, and the synthesis of nascent peptide chains was detected in ME49-Δku80 and ME49-Δeno1 strains using puromycin labeling and Western blotting assay to examine the effect of eno1 deletion on the synthesis of T. gondii proteins. Results The eno1-deleted strain ME49-Δeno1 and its two complementary strains ME49-Δeno1-Comp-eno1 and ME49-Δeno1-Comp-eno2 were successfully generated. Plaque, proliferation, and invasion assays showed that the areas of plaques were (16 538 ± 14 310), (4 376 ± 5 355), (17 377 ± 14 333), and (8 710 ± 8 207) μm², and the average numbers of parasites per parasitophorous vacuole were (5.836 ± 0.382), (4.792 ± 0.150), (5.165 ± 1.243), and (3.872 ± 0.301) in ME49-Δku80, ME49-Δeno1, ME49-Δeno1-Comp-eno1, and ME49-Δeno1-Comp-eno2 strains, while the invasion efficiencies of ME49-Δku80, ME49-Δeno1, ME49-Δeno1-Comp-eno1, and ME49-Δeno1-Comp-eno2 strains were (57.802 ± 6.932)%, (19.679 ± 4.508)%, (63.833 ± 1.198)%, and (35.901 ± 8.098)%, respectively, indicating that deletion of eno1 inhibited the growth (F = 38.290, P < 0.01), intracellular proliferation (F = 4.467, P < 0.05), and invasion ability (F = 36.650, P < 0.01) of T. gondii. Complementation of eno1 fully restored these abilities, and there were no significant differences between ME49-Δeno1-Comp-eno1 and ME49-Δku80 in terms of the plaque areas (t = 0.459, P > 0.05), average number of T. gondii tachyzoites per parasitophorous vacuole (t = 0.895, P > 0.05), or invasion efficiency (t = 1.485, P > 0.05). Nevertheless, complementation of eno2 partially restored the growth, proliferation and invasion abilities of T. gondii. The area of plaques generated by the ME49-Δeno1-Comp-eno2 strain was larger than that by the ME49-Δeno1 strain (t = 5.263, P < 0.05), but smaller than that by the ME49-Δku80 strain (t = 4.905, P < 0.01), and the average number of tachyzoites per parasitophorous vacuole was lower in the ME49-Δeno1-Comp-eno2 strain than in the ME49-Δku80 strain (t = 6.998, P < 0.05), while the invasion efficiency of the ME49-Δeno1-Comp-eno2 strain was higher than that of the ME49-Δeno1 strain (t = 3.032, P < 0.05) but lower than that of the ME49-Δku80 strain (t = 3.559, P < 0.05). Following alkaline induction for 48 h, there was no significant difference in the relative transcriptional level of sag1 gene between the ME49-Δeno1 strain (1.067 ± 0.115) and the ME49-Δku80 strain (1.002 ± 0.154) (t = 0.584, P > 0.05), and the relative transcriptional level of bag1 gene was lower in the ME49-Δeno1 strain (13.172 ± 1.679) than in the ME49-Δku80 strain (20.338 ± 1.344) (t = 5.770, P < 0.01). Following alkaline induction for 96 h, the relative transcriptional level of sag1 gene was higher in the ME49-Δeno1 strain (0.957 ± 0.132) than in the ME49-Δku80 strain (0.727 ± 0.053) (t = 2.803, P < 0.05), and the relative transcriptional level of bag1 gene was lower in the ME49-Δeno1 strain (162.248 ± 23.377) than in the ME49-Δku80 strain (231.413 ± 12.910) (t = 4.486, P < 0.05). Western blotting assay detected no significant difference in the level of nascent peptide synthesis between the ME49-Δku80 and ME49-Δeno1 strain, indicating no significant effect of eno1 deletion on the synthesis ability of T. gondii proteins. Conclusion Bradyzoite-specific enolase eno1 promotes the growth of T. gondii tachyzoites and positively regulates the transition from tachyzoites to bradyzoites. It is a key regulator but not compulsory in the T. gondii tachyzoite-bradyzoite transition. In addition, ENO1 cannot be functionally replaced by ENO2 in the proliferation of T. gondii tachyzoites.

Key words: Toxoplasma gondii, Enolase 1, Enolase 2, Glycolysis, Tachyzoite-bradyzoite interconversion