Upregulation of the lysosomal trafficking regulator gene (<i>LYST/Beige</i>) in macrophages infected with <i>Leishmania amazonensis</i> modulates parasitophorous vacuole expansion and parasite intracellular growth.

<p>(A) Upregulation of <i>LYST/Beige</i> transcripts in BMM infected with <i>L. amazonensis</i>. BMM were infected for 30 min with axenic amastigotes, washed and incubated for the indicated periods, followed by RNA extraction and quantitative real-time PCR (qPCR) analysis. The data represents the mean +/− SD of triplicate determinations. Asterisks indicate significant differences from non-infected samples (NI): ** <i>P</i><0.005 (Student t test). (B) Increased PV expansion in <i>LYST/Beige</i>-deficient BMM (bg^J/bg^J). BMM were infected for 30 min with axenic amastigotes and the size of PVs was determined microscopically after the indicated periods. The values represent the mean of 50 independent PV measurements, asterisks indicate significant differences from the same time points in wild type BMM: * <i>P</i><0.05, ** <i>P</i><0.01 (Student t test). (C) Wild type and bg^J/bg^J BMM infected with <i>L. amazonensis</i> were fixed immediately or after 48–72 h, and stained with anti-Lamp1 mAb (green) and DAPI (DNA, blue) stain. Arrows point to heterogeneously sized Lamp1-positive PVs in bg^J/bg^J BMM. Bar = 15 µm. (D) Phase-contrast images showing higher magnifications of parasite-containing PVs (arrows) in wild type or bg^J/bg^J BMM 48 h after infection. (Bar = 5 µm). (E) <i>L. amazonensis</i> intracellular growth is enhanced in bg^J/bg^J BMM. BMM were infected for 60 min and the number of intracellular parasites was determined after the indicated periods. The data (expressed as fold increase in parasite numbers over the 60 min values) corresponds to the mean +/− SD of triplicates. Asterisks indicate significant differences from the corresponding time points in wild type BMM: * <i>P</i><0.05 (Student t test).