用户名： 密码： 验证码：
Rac1's Regulation on Host Cell Cytoskeleton Reorganization affects Toxoplasma gondii Invasion Efficiency
详细信息    查看官网全文
摘要
Objectives: Rac1 is a small GTPase in Rho family and plays an important role in the cytoskeleton reorganization. It was previously reported that Rac1 was activated and concentrated on the parasitophorous vacuole membrane(PVM) during the invasion of T. gondii tachyzoites into the host cell. The role of the host cell Rac1 in T. gondii invasion was further investigated in this research. Methods:HFF cells were either treated with Rac1 inhibitor(NSC 23766) or left untreated for control, and then infected with T. gondii tachyzoites for 30 minutes, then followed by paraformaldehyde fixing and Giemsa staining. The invasion rates of T. gondii were counted and the positions of the invaded tachyzoites were observed under a phase contrast microscope. Immune fluorescence(IF) of host cell Rac1 and actin in the inhibitor treated or untreated cells were observed to show the different cytoskeleton reorganization. The co-localization of host cell Rac1 with T. gondii ROP2 was observed in the T. gondii infected cells under a fluorescence microscope, after the host cell Rac1 and Tg ROP2 were labeled with different immune fluorescence. The phosphorylation level of Rac1 in HFF cells without infection or infected with T. gondii for different time was detected by western blot. Results: T. gondii infection rate of the normal cells was significantly higher than which of Rac1 inhibitor treated cells(p <0.01). Most(about 83%) of the invaded tachyzoites only stayed in the peri-membrane region of the host cell in Rac1 inhibitor treated group, while only about 17% of the invaded tachyzoites can enter deeply into the host cell, even enter the nuclei in the untreated group. The IF observation for host cell Rac1 and actin showed that cytoskeleton reorganization was more active in the untreated cells comparing to the cells treated with Rac1 inhibitor. The endogenous host cell Rac1 co-localized with T. gondii ROP2 on the PVM of the newly invaded parasites, and then Rac1 faded away from the PVM when the parasites entering deeply into the host cell. This process probably related to Rac1 cycling from the cytoplasm to the PVM or reversely from PVM to the cytoplasm. The phosphorylation level of host cell Rac1 increased with the infection time(0~6 hours) by T. gondii. It can be deduced that Rac1 inhibitor hindered T. gondii invasion, probably through inhibiting the host cell cytoskeleton reorganization. The Rac1 GTPase phosphorylation may help to the deactivation of the Rac1 GTPase and being released from the PVM to the cytoplasm.Conclusion: Rac1 regulates host cell cytoskeleton reorganization, and through which affects Toxoplasma gondii invasion efficiency. The endogenous host cell Rac1 co-localized with T. gondii ROP2 on the PVM during early stage of infection, and Rac1 disappeared from the PVM when the parasites entered deeply into the host cell. Rac1 phosphorylation levels in host cells increased with T. gondii infection time and may related to the disappearance of the Rac1 from the PVM.
Objectives: Rac1 is a small GTPase in Rho family and plays an important role in the cytoskeleton reorganization. It was previously reported that Rac1 was activated and concentrated on the parasitophorous vacuole membrane(PVM) during the invasion of T. gondii tachyzoites into the host cell. The role of the host cell Rac1 in T. gondii invasion was further investigated in this research. Methods:HFF cells were either treated with Rac1 inhibitor(NSC 23766) or left untreated for control, and then infected with T. gondii tachyzoites for 30 minutes, then followed by paraformaldehyde fixing and Giemsa staining. The invasion rates of T. gondii were counted and the positions of the invaded tachyzoites were observed under a phase contrast microscope. Immune fluorescence(IF) of host cell Rac1 and actin in the inhibitor treated or untreated cells were observed to show the different cytoskeleton reorganization. The co-localization of host cell Rac1 with T. gondii ROP2 was observed in the T. gondii infected cells under a fluorescence microscope, after the host cell Rac1 and Tg ROP2 were labeled with different immune fluorescence. The phosphorylation level of Rac1 in HFF cells without infection or infected with T. gondii for different time was detected by western blot. Results: T. gondii infection rate of the normal cells was significantly higher than which of Rac1 inhibitor treated cells(p <0.01). Most(about 83%) of the invaded tachyzoites only stayed in the peri-membrane region of the host cell in Rac1 inhibitor treated group, while only about 17% of the invaded tachyzoites can enter deeply into the host cell, even enter the nuclei in the untreated group. The IF observation for host cell Rac1 and actin showed that cytoskeleton reorganization was more active in the untreated cells comparing to the cells treated with Rac1 inhibitor. The endogenous host cell Rac1 co-localized with T. gondii ROP2 on the PVM of the newly invaded parasites, and then Rac1 faded away from the PVM when the parasites entering deeply into the host cell. This process probably related to Rac1 cycling from the cytoplasm to the PVM or reversely from PVM to the cytoplasm. The phosphorylation level of host cell Rac1 increased with the infection time(0~6 hours) by T. gondii. It can be deduced that Rac1 inhibitor hindered T. gondii invasion, probably through inhibiting the host cell cytoskeleton reorganization. The Rac1 GTPase phosphorylation may help to the deactivation of the Rac1 GTPase and being released from the PVM to the cytoplasm.Conclusion: Rac1 regulates host cell cytoskeleton reorganization, and through which affects Toxoplasma gondii invasion efficiency. The endogenous host cell Rac1 co-localized with T. gondii ROP2 on the PVM during early stage of infection, and Rac1 disappeared from the PVM when the parasites entered deeply into the host cell. Rac1 phosphorylation levels in host cells increased with T. gondii infection time and may related to the disappearance of the Rac1 from the PVM.
引文

© 2004-2018 中国地质图书馆版权所有 京ICP备05064691号 京公网安备11010802017129号

地址：北京市海淀区学院路29号 邮编：100083

电话：办公室：(+86 10)66554848；文献借阅、咨询服务、科技查新：66554700