Figure 5.

Episomal expression of γGCS-(HA)₃ leads to increased BSO resistance. A. Southern blot analysis of wild-type D10 parasites (lane 1) in comparison with D10^γGCS transfected with expression plasmid pCHD-γgcs(HA)₃ (lane 2). The 3.9 kb band corresponding to endogenous γgcs and the 9.9 kb band diagnostic for the plasmid are indicated. B. Western blot analysis with anti-(HA)₃ antibody (mouse, 1:1000) confirmed expression of γGCS-(HA)₃ in D10^γGCS. As a loading control an anti-P. falciparum 1-Cys peroxiredoxin antibody (α-TSAI, rabbit 1:120 000) was used. C. The BSO IC₅₀ for wild-type D10 (○) was significantly lower (58.0 ± 2.2 µM) than for D10^γGCS (▴, 111.2 ± 11.1 µM) parasites expressing γGCS-(HA)₃ (P < 0.001, unpaired Student's t-test). Data represent means of three independent determinations, each done in duplicate. Error bars represent SD. D. GSH levels in D10 and D10^γGCS under normal growth conditions and after incubation with 50 µM BSO for 2 h. Results represent means ± SEM of 3 to 5 independent determinations. There were no significant differences of GSH levels found between D10 (832 ± 50 nmol/10¹⁰ cells) and D10^γGCS parasites (757 ± 85 nmol/10¹⁰ cells) grown in normal culture medium. Exposure to BSO significantly decreased GSH levels in both D10 and D10^γGCS to 138 ± 41 and 199 ± 74 nmol/10¹⁰ cells respectively. Statistical analyses were performed using one-way ANOVA and Newman–Keuls post test (***P < 0.001).