Examining growth of B divergens in different RBC genotypes at different O2 concentrations.B divergens was grown synchronously in vitro at 1%, 5%, 10%, and 21% O2, with constant 5% CO2 and remaining N2. Three genotypes were examined: (A) AA wild type, (B) AS heterozygous for SCD and (C) sickle (SS) homozygous for SCD. Parasitemia at day 3 after invasion were plotted as the percentage of enhancement or inhibition compared with its own growth at standard 5% O2 used widely for in vitro cultures. As evident in (A), parasites grown at 1% O2 in AA cells showed similar growth as 5% O2 (moderate, nonsignificant inhibition of 10.05% ± 5.97%). At 10% and 21% O2, the growth was enhanced (1.39% ± 1.37%, P = .1559 and 13.91% ± 8.41%, P = .0467, respectively). For parasites growing in AS RBCs (B), there was a significant inhibition of growth at 1% O2 (26.48% ± 8.27%). At 5% O2, AA and AS growth rates were comparable. Similar to AA cells, AS cells exhibited enhanced growth at 10% (25.38% ± 7.23%) and 21% O2 (47.9% ± 7.96%). (C) For the SS genotype, at 1% O2, parasites were inhibited almost unchanged as compared with 5% O2 at which parasites also suffered from impaired growth. However, at higher O2 levels, parasites grew better, as shown by a 31.34% ± 5.03% and 101.29% ± 21.46% enhancement of growth compared with 5% at 10% and 21%, respectively. (D) The percentage of parasitemia is plotted for AA and SS cells grown at 21% O2 over days after the invasion. As shown, starting from day 2, AA cells support better growth of parasite, and this difference is evident at day 3, when parasites in AA cells reach their peak, in vitro parasitemia of 48.423% ± 4.105%, whereas parasites in the SS cells are at 29.458% ± 6.337% parasitemia. On day 4, the AA culture crashed (as shown by #), whereas the SS culture reached its peak of 45.367% ± 4.866%. (E and F) Transfer experiments: parasites were grown at 1% O2 (E) and 5% O2 (F) and on day 5 after the invasion at parasitemia of 7.87% ± 0.948% and 9.053% ± 1.6%, respectively, were transferred to 21% O2. After transfer, parasites previously grown at 1% and 5% reached parasitemia of 32.27% ± 9.843% and 34.848% ± 9.180%, respectively. Thus, after stagnation of growth at low- O2 conditions, transfer to a higher O2 condition can rescue the growth of the parasites to near-normal levels in SS host RBCs. (G) We examined whether parasite invasion was affected by different O2 levels across the 3 genotypes. No significant differences were found (P = .4512), thus suggesting that the invasion of B divergens remains similar across genotypes and at different O2 culture conditions.
