Figure 6. Successful invasion is required for upregulation of UPRT promoter activity.

(A) Myc-B treated parasites attach to host cells and secrete micronemes and rhoptries but do not invade. A parasite clone stably expressing UPRT-FLU and TUB-RLU (UT-DualLuc) was treated with Myc-B and added onto HFF cells in an 8 well chamber slide. After 30 min, slides were fixed and stained for M2AP without permeabilization. The slides were then treated with saponin, which permeabilizes the host cell plasma membrane but not the parasite cell membrane, and then stained for ROP1.

(B) UT-DualLuc parasites were treated with DMSO (vehicle control) or Myc-B and added to HFF cells for 30 min. Non-attached parasites were gently washed and the cultures were incubated at 37°C for 2 h followed by assessment of luciferase activity as described in experimental procedures. UPRT-FLU is not upregulated in Myc-B treated parasites indicating that the activation of signaling pathways associated with microneme or rhoptry discharge are not sufficient to induce expression reprogramming. n=3 independent experiments, each with triplicate samples. Error bars, SEM.

(C) Fresh naturally egressed RH parasites were purified, treated with DMSO (vehicle control) or Myc-B and added onto HFF cells. After incubation at 37°C for 30 min, Myc-B treated parasites were gently washed once to remove unattached parasites while DMSO treated parasites were vigorously washed thrice to remove non-attached and non-invaded parasites and the cultures were further incubated at 37°C for 2 h. DMSO treated parasites were incubated without HFF cells for 2 h and 30 min to serve as extracellular parasites. RNA was purified from pellets of extracellular parasites, HFF cells with only attached parasites (Myc-B treated), and 2 h intracellular parasites (DMSO treated) and analyzed by qRT-PCR. n=3 independent experiments. Error bars, SEM.