Toxoplasma Gondii ROP17 Promotes Autophagy via the Bcl-2–Beclin 1 Pathway
Authors: Guo Min , Sun Jia , Wang Wen-tao , Liu Yue-hua , Qin Ke-ru , Meng Tao-tao , Hu Jin-rui , Li Xin-yang , Xu Bai-xue , Meng Xiao-li , Liu Juan-juan , Liu Hong-li , Wang Wei , Guo Hai-xiu , Chen Zhao-yang , Wang Chun-fang , Wang Hailong 
Published: July 2020

Abstract
Background
Toxoplasmosis caused by Toxoplasma gondii (T. gondii) has been and continues to be a major threat to public health worldwide. Proteins with serine/threonine (S/T) kinase activity, such as rhoptry organelle protein (ROP)16 and ROP18, are secreted by T. gondii rhoptries organelles that play important roles in modulating the survival and proliferation of Toxoplasma. ROP17 is another important effector kinase protein. Nevertheless, little is known about its function in regulating the interplay between T. gondii and host cells during infection. In this study, we investigated the function and the underlying mechanisms of ROP17 in vitro and in vivo.
Methods
The autophagy of cells in the small intestines of mice infected with T. gondii tachyzoite (RH strain) was detected by assessing the LC3B, Beclin 1 and p62 levels using immunohistochemical staining. ROP17 overexpression augmented starvation-induced autophagy in HEK 293T cells as measured by MDC staining, transmission electron microscopy (TEM), fluorescence microscopy and Western blot analysis. The interaction of ROP17 and Bcl-2 was confirmed using co-immunoprecipitation (co-IP) analysis. Moreover, the levels of phosphorylation of Bcl-2 and total Bcl-2 in the small intestines of mice infected with T. gondii tachyzoite were detected using immunohistochemical staining. In the end, Pearson coefficient was used to analyse correlations between the expression of ROP17 with the expression of autophagy markers LC3B, beclin 1 and p62, and with phosphorylation of Bcl-2 and Bcl-2.
Results
Infection with T. gondii resulted in autophagy of mouse small intestine cells accompanied by increased levels of LC3B and Beclin 1 and decreased levels of p62, which were measured by immunohistochemistry. Meantime, the elevated levels of Phosphorylated Bcl-2 and declined Bcl-2 in T. gondii-infected mouse small intestine tissue and in ROP17 overexpressing HEK 293T cells were detected by immunohistochemistry and Western blotting. ROP17 overexpression promoted serum starvation-induced cellular autophagy in the HEK293T cells, reflecting the augmentation of autophagosomes and GFP-LC3B puncta accompanied by increased levels of LC3B and Beclin 1 and decreased levels of p62. Moreover, we found that ROP17 interacted with Bcl-2 and phosphorylated and degraded it to liberate Beclin 1, thus promoting autophagy. Pearson coefficient analysis showed that there exsited strong positive correlations between the expression of ROP17 with the expression of LC3B, Beclin 1 and phosphorylation of Bcl-2, while strong negative correlations between the expression of ROP17 and the expression of p62 and Bcl-2.
Conclusions
Our findings indicate that ROP17 plays a pivotal role in maintaining Toxoplasma survival and proliferation in host cells by promoting cellular autophagy, which depends on the Beclin 1-Bcl-2 pathway.