However, the dominant negative mutants RhoA-N19 and Rac1-N17 overexpressed in COS-7 cells inhibited the cell invasion by T. gondii tachyzoites significantly; the infection rates were approximately 60% of that of the mock cells (p < 0.01) (Figure 7A-B, respectively). Silencing RhoA, Rac1 or both RhoA and Rac1 in 16-HBE cells also showed a significant inhibition of cell invasion by tachyzoites (p < 0.01) Figure 7C-E). The infection rates of RhoA and Rac1 silenced Selleck Pifithrin �� cells were about 65% of that of the mock cells, while the infection rate of RhoA and Rac2 double-silenced cells was about 50% of that of the mock cells (Figure 7C). Figure 7 The overexpression of dominant negative mutants of Rho GTPases and the expression silencing

Oligomycin A of Rho GTPases in host

cells diminished the invasiveness of T. gondii RH tachyzoites. (A-B) RhoA or Rac1 overexpression: When compared with the untransfected cells (mock group), RhoA-WT or Rac1-WT overexpressed cells showed the almost same infection rate, while dominant-negative mutant RhoA-N19 or Rac1 N17 overexpressed cells showed a significantly lower infection rate (P = 0.001 and P = 0.005), proximately 60% of the Mock. (C) Silencing of RhoA or Rac1: When compared with the untransfected cells (mock group) and negative control siRNA transfected groups, cells transfected with RhoA siRNA, Rac1 siRNA or RhoA + Rac1 siRNA showed a significantly lower infection rate (P < 0.001). It was about 65% of the Mock in the two single knockdown groups and about 50% of the Mock in the double knockdown group. (D-E)

Detection of RhoA or Rac1 RNAi efficiency: anti-actin panel showed the same amount of total protein was loaded for detection in different cell lysates including mock, negative control siRNA, RhoA or RAC1 siRNA, and RhoA + Rac1 siRNA transfected groups. Anti-RhoA panel showed the apparent inhibition of RhoA expression in RhoA silenced and RhoA + Rac1 silenced cells; anti-Rac1 panel showed the apparent inhibition of Rac1 expression in Rac1 and RhoA + Rac1 silenced cells. Discussion The function of the Rho and Rac GTPases accumulated on PVM Immunity-related GTPases (IRGs) also known for as p47 GTPases, are key mediators of interferon-gamma-induced resistance to pathogens [19]. They cycle between GDP-GTP bound forms, and cooperatively oligomerize in the GTP-bound conformation on the T. gondii PVM [20]. Sequential recruitment of multiple IRGs to the PVM results in disruption of PVM and parasite digestion within 2 hr of infection [21]. Virulent type I see more strains resist recruitment and avoid clearance, while less virulent type II and III strains are effectively cleared by IRGs [22]. It was reported that a serine threonine kinase secreted by T. gondii, ROP18, binds to and phosphorylates IRGs on the PVM, and the phosphorylation of IRGs prevented clearance of T. gondii within inflammatory monocytes and IFN-γ-activated macrophages, conferring parasite survival in vivo[23].