L. major GP63 cleaves VAMP8 and prevents its recruitment to phagosomes.

(A) VAMP8 cleavage in C57BL/6 x 129 BMM infected for 2 h with opsonized WT, Δgp63 or Δgp63+gp63 L. major NIH S promastigotes was assessed by immunoblot analysis. VAMP8 band intensities were measured by spot densitometry, normalized to the β-actin loading control and compared to the uninfected control (Ctl) cells. (B) Confocal microscopy images of C57BL/6 x 129 BMM fed zymosan particles or infected with opsonized WT, Δgp63, or Δgp63+gp63 L. major NIH S promastigotes for 1 h. VAMP8 is in green; nuclei are in blue. Asterisks indicate phagosomes containing zymosan particles, white arrows indicate parasite nuclei, and red-filled arrowheads point to VAMP8 recruitment. Scale bar, 5 μM. (C) Quantification of VAMP8-positive phagosomes at 1 and 2 h after infection. Data are presented as the mean ± SEM of values from triplicate samples of an experiment representative of more than three others. *p<0.05, **p<0.01.