Heme deprivation increases LHR1 expression and promotes uptake of the heme analog ZnMP by L. amazonensis promastigotes.

(A) qPCR quantification of LHR1 transcripts relative to the reference gene GAPDH, in log phase L. amazonensis promastigotes grown in medium with or without heme for 15 h. The results represent the mean+/−standard deviation (SD) of three independent experiments. (B) Confocal fluorescence images of L. amazonensis promastigotes cultured for 15 h with or without heme, incubated with ZnMP for 3 or 6 h, and imaged live under identical conditions. ZnMP accumulation within the parasites is shown in red, and the viability dye FDA is shown in green. (C) Immunoblot of extracts of L. amazonensis promastigotes expressing LHR1 tagged or not with the 3xFLAG epitope, and probed with anti-FLAG M2 monoclonal antibodies. (D) Flow cytometry quantification of ZnMP uptake by promastigotes untransfected (top) or transfected with LHR1 (bottom), after 15 h pre-incubation in the presence or absence of heme, followed by uptake under the same conditions. The numbers indicate the percentage of cells with fluorescence levels above the gate value (vertical line on horizontal bar, determined from measurements on parasites not incubated with ZnMP – grey shaded profile).