Re-expression of amastin sequences in RNAi knockdown parasites rescue infection capacity of <i>L</i>. <i>braziliensis</i>.

<p>(A) Total protein extracts from promastigotes (P) and amastigotes (A) of WT <i>L</i>. <i>braziliensis</i>, the cloned cell RNAi-1060 cl1 and two cloned cell lines derived from the RNAi-1060 parasites the were transfected with an RNAi-resistant amastin gene (RNAi-1060-R2 and RNAi-1060-R4) were analysed by western blot using an anti-HA antibody. Bands corresponding to the Ama1060 synthetic gene containing the HA epitope are shown only in the re-expressor cell lines. The same blot was incubated with anti-tubulin antibody as a loading control. (B) Stationary phase promastigotes from WT <i>L</i>. <i>braziliensis</i>, two cloned cell lines expressing amastin dsRNA, RNAi-1060 cl1 and cl5 and two cloned cell lines that express a RNAi resistant amastin sequence were used to infect BALB/c mice peritoneal macrophages at a ratio of 10:1 (parasites/cell) and the numbers of intracellular amastigotes determined 24 and 72 hours post-infection. (C) Stationary phase promastigotes (10⁷ parasites) from WT <i>L</i>. <i>braziliensis</i>, <i>L</i>. <i>braziliensis</i> transfected with the pIR1PHLEO vector containing GFP, the two cloned cell lines expressing amastin dsRNA, RNAi-1060 cl1 and cl5 and two cloned cell lines that express a RNAi resistant amastin sequence were used to infected BALB/c mice footpads. One or two weeks post-infection, parasitism was evaluated by the limiting dilution of parasites recovered from the mice footpads.