Enzymatically active Rho and Rac small-GTPases are involved in the establishment of the vacuolar membrane after Toxoplasma gondii invasion of host cells

Abstract

BackgroundGTPases 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.ResultsIn 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.ConclusionsThe 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.

Genome-Wide CRISPR Screen Identifies Host Factors Required by Toxoplasma gondii Infection

“…After that, T. gondii recruits host cell microtubules to form conduits, along which host organelles are transported to the PV (Coppens et al, 2006). The RacGTPases were activated upon T. gondii invasion, and when F-actin polymerization was inhibited in NSC23766-treated cells, the efficiency of T. gondii attachment, invasion, and replication was significantly decreased (Na et al, 2013;Wei et al, 2019). Furthermore, it has been reported that six human proteins (PTK9L, MAPK7, PHPT1, MYLIP, PTPRR, and PPIL2) facilitate T. gondii entry by modifying host actin dynamics (Gaji et al, 2013).…”

“…For example, as T. gondii lacks the enzymes for polyamine, arginine, and purine biosynthesis, the intracellular parasites have to get these nutrients from their host cells (Schwartzman and Pfefferkorn, 1982;Fox et al, 2004;Cook et al, 2007). We previously reported that host RhoA and Rac1 GTPases were activated upon T. gondii infection and facilitated T. gondii invasion (Na et al, 2013;Wei et al, 2019). It also has been reported that host hypoxia-inducible transcription factor 1 (HIF-1)/hexokinase 2 (HK2) can reprogram the host cell's metabolism to create an environment conducive for parasite replication at 3% physiological oxygen levels (Menendez et al, 2015).…”

Phosphoproteome of Toxoplasma gondii Infected Host Cells Reveals Specific Cellular Processes Predominating in Different Phases of Infection

“…26 The small GTPases, including Rho, Rac, and adenosine diphosphate-ribosylation factor 6, have been reported to accumulate on the PVM and function in host cell cytoskeleton reorganization during T. gondii invasion, which facilitates PV formation and enlargement in the host cell. 13,14 Interestingly, the GO term "cytoskeletal protein binding" was enriched in all comparisons (M versus L, H versus L, and H versus M), which suggested that host cell cytoskeletal FIGURE 3. Gene ontology (GO) enrichment analysis of the 892 identified phosphoproteins.…”

“…Studies have shown that the maintenance of host cell actin cytoskeleton integrity is important for parasite invasion and successful penetration of host cells by apicomplexans; during this process, significant host cell cytoskeletal reorganization is accompanied. 13,14,27 Toxoplasma gondii infection induces and maintains a "proliferation response" including dysregulation of the cell cycle in infected host cells. This response may fulfill critical growth and multiplication requirements of the parasite during intracellular residence.…”

“…5,6 Phosphorylation turns many enzymes on and off, thereby altering their functions and activities, and plays a significant role in a wide range of cellular processes. 6 Toxoplasma gondii infection triggers extensive host cell signal transduction involved in the regulation of antiapoptosis, 7 apoptosis, 8,9 cell immunity, 10 cell-cycle control, 11 calcium regulation, 12 cytoskeletal reorganization, 13,14 and all other cellular processes. Phosphoproteomic analysis of Plasmodium falciparum schizonts and T. gondii tachyzoites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) revealed a large number of unknown phosphorylation sites in both parasites; this analysis also provided abundant clues for the role of phosphorylation in host-pathogen interactions.…”