Key Laboratory of Prevention and Control for Emerging Infectious Diseases of Guangdong Higher Institutes, Department of Pathogen Biology, School of Public Health and Tropical Medicine, Southern Medical University, Guangzhou, Guangdong 510515, China.
BMC Microbiol. 2013 May 30;13:125. doi: 10.1186/1471-2180-13-125.
GTPases are the family of hydrolases that bind and hydrolyze guanosine triphosphate. The large Immunity-related GTPases and the small GTPase ADP-ribosylation factor-6 in host cells are known to accumulate on the parasitophorous vacuole membrane (PVM) of Toxoplasma gondii and play critical roles in this parasite infection, but these GTPases cannot explain the full extent of infection.
In this research, RhoA and Rac1 GTPases from the host cell were found to accumulate on the PVM regardless of the virulence of the T. gondii strains after T. gondii invasion, and this accumulation was dependent on their GTPase activity. The real-time micrography of T. gondii tachyzoites invading COS-7 cells overexpressing CFP-RhoA showed that this GTPase was recruited to the PVM at the very beginning of the invasion through the host cell membrane or from the cytosol. Host cell RhoA and Rac1 were also activated after T. gondii tachyzoites invasion, which was needed for host cell cytoskeleton reorganization to facilitate intracellular pathogens invasion. The decisive domains for the RhoA accumulation on the PVM included the GTP/Mg2+ binding site, the mDia effector interaction site, the G1 box, the G2 box and the G5 box, respectively, which were related to the binding of GTP for enzymatic activity and mDia for the regulation of microtubules. The recruited CFP-RhoA on the PVM could not be activated by epithelial growth factor (EGF) and no translocation was observed, unlike the unassociated RhoA in the host cell cytosol that migrated to the cell membrane towards the EGF activation spot. This result supported the hypothesis that the recruited RhoA or Rac1 on the PVM were in the GTP-bound active form. Wild-type RhoA or Rac1 overexpressed cells had almost the same infection rates by T. gondii as the mock-treated cells, while RhoA-N19 or Rac1-N17 transfected cells and RhoA, Rac1 or RhoA + Rac1 siRNA-treated cells showed significantly diminished infection rates compared to mock cells.
The accumulation of the RhoA and Rac1 on the PVM and the requisite of their normal GTPase activity for efficient invasion implied their involvement and function in T. gondii invasion.
GTPases 是一类水解酶，能够结合并水解鸟苷三磷酸。宿主细胞中的大免疫相关 GTPases 和小 GTPase ADP-核糖基化因子-6 已知会聚集在刚地弓形虫的寄生泡膜（PVM）上，并在寄生虫感染中发挥关键作用，但这些 GTPases 并不能解释感染的全部程度。
在这项研究中，无论刚地弓形虫株的毒力如何，宿主细胞中的 RhoA 和 Rac1 GTPases 在刚地弓形虫入侵后都会聚集在 PVM 上，这种聚集依赖于它们的 GTPase 活性。实时显微镜观察显示，在 COS-7 细胞中转染 CFP-RhoA 后，速殖子入侵时，该 GTPase 通过宿主细胞膜或细胞质被招募到 PVM 上。刚地弓形虫速殖子入侵后，宿主细胞 RhoA 和 Rac1 也被激活，这对于宿主细胞细胞骨架重组以促进细胞内病原体入侵是必要的。RhoA 在 PVM 上的聚集的决定性结构域包括 GTP/Mg2+结合位点、mDia 效应子相互作用位点、G1 盒、G2 盒和 G5 盒，分别与酶活性的 GTP 结合和微管的 mDia 调节有关。在 PVM 上募集的 CFP-RhoA 不能被表皮生长因子（EGF）激活，也没有观察到易位，而宿主细胞质溶胶中未关联的 RhoA 则向 EGF 激活点迁移到细胞膜，这与 RhoA 在 PVM 上募集的 RhoA 或 Rac1 处于 GTP 结合的活性形式的假说一致。与 mock 处理的细胞相比，过表达野生型 RhoA 或 Rac1 的细胞感染刚地弓形虫的比率几乎相同，而过表达 RhoA-N19 或 Rac1-N17 的细胞以及 RhoA、Rac1 或 RhoA+Rac1 siRNA 处理的细胞的感染率明显低于 mock 细胞。
RhoA 和 Rac1 在 PVM 上的聚集以及它们正常 GTPase 活性对有效入侵的必要性表明它们参与并在刚地弓形虫入侵中发挥作用。
