Deletion of the Acidic Domain abolishes the functional effect of ALT transfection. (A-C) Immunofluorescent detection of ADD (acidic domain deleted mutant of ALT-2) protein. Paraformaldehyde-fixed wild-type promastigotes (A) and cloned ADD transfected parasites (B) were incubated with mouse antiserum to anti-ALT-2, and detected with an anti-mouse-FITC secondary antibody. (C) Cloned ADD transfected parasites were also incubated with normal mouse serum. (D) Bone marrow-derived macrophages were infected with stationary phase amastigotes of wild-type L. mexicana or transfected alt-2 and ADD parasites at a ratio of 10 parasites per cell. After 7 days, cells were stained with Giemsa. The percentage of macrophages infected with parasites was determined by counting 3 samples (from 3 different wells) of 100 cells. The χ2 test showed significant differences in the number of infected macrophages between wild-type and alt-2 transfectants (p < 0.001), but not between wild-type and add-transfected parasites.