Figure 5.

Proper regulation of the pfsr1 gene is essential for parasites’ proliferation in human RBCs. Over-expression of pfsr1 gene inhibits parasite proliferation. (A) Schematic map of the plasmid (pHBIPfSR1myc) used for ectopically over-expression using increasing concentrations of blasticidin as described (28). Steady-state mRNA levels of pfsr1 and renilla luciferase genes were measured using qRT–PCR from tightly synchronized parasite cultures 36-h post-invasion (hpi). Values are presented as relative copy number to the housekeeping genes arginyl-tRNA synthetase (PFL0900c). (B) Transcription levels of pfsr1 and renilla luciferase expressed from the transfected episomes by parasites growing under 2 and 6 µg/ml blasticidin selection. (C) Growth curves of the parasite populations over-expressing pfsr1 (light gray) compared with those that similarly express renilla luciferase (black) from the pHBIPfSR1myc and the pHBIRH plasmids, respectively, under 2 μg/ml blasticidin. Arrows indicate days in which the culture was cut down to avoid over-parasitemia. Each curve represents the average of three different cultures grown in parallel. (D) Growth curves of the parasite populations over-expressing pfsr1 (light gray) compared with those that similarly express renilla luciferase (black) from the pHBIPfSR1myc and the pHBIRH plasmids, respectively, under 6 μg/ml blasticidin. Day 0 represents the day in which blasticidin concentration was increased. Each curve represents the average of three different parasitemia measurements. (E) Growth curves of the parasite populations over-expressing the different PfSR1 mutants (gray) compared with those that similarly express renilla luciferase (black) under 2 μg/ml blasticidin.