Leishmania major Promastigotes Evade LC3-Associated Phagocytosis through the Action of GP63.

The protozoan Leishmania parasitizes macrophages and evades the microbicidal consequences of phagocytosis through the inhibition of phagolysosome biogenesis. In this study, we investigated the impact of this parasite on LC3-associated phagocytosis, a non-canonical autophagic process that enhances phagosome maturation and functions. We show that whereas internalization of L. major promastigotes by macrophages promoted LC3 lipidation, recruitment of LC3 to phagosomes was inhibited through the action of the parasite surface metalloprotease GP63. Reactive oxygen species generated by the NOX2 NADPH oxidase are necessary for LC3-associated phagocytosis. We found that L. major promastigotes prevented, in a GP63-dependent manner, the recruitment of NOX2 to phagosomes through a mechanism that does not involve NOX2 cleavage. Moreover, we found that the SNARE protein VAMP8, which regulates phagosomal assembly of the NADPH oxidase NOX2, was down-modulated by GP63. In the absence of VAMP8, recruitment of LC3 to phagosomes containing GP63-deficient parasites was inhibited, indicating that VAMP8 is involved in the phagosomal recruitment of LC3. These findings reveal a role for VAMP8 in LC3-associated phagocytosis and highlight a novel mechanism exploited by L. major promastigotes to interfere with the host antimicrobial machinery

VAMP8 is essential for LC3 recruitment to <i>L</i>. <i>major</i> parasitophorous vacuoles.

<p>(A) Confocal microscopy images of WT or <i>Vamp8</i>^-/- C57BL/6 x 129 BMM infected for 1 h with opsonized WT, Δ<i>gp63</i>, or Δ<i>gp63</i>+<i>gp63 L</i>. <i>major</i> NIH S promastigotes. VAMP8 is in green; nuclei are in blue. White arrows indicate parasite nuclei, and red-filled arrowheads point to VAMP8 recruitment. Scale bar, 5 μM. (B) Quantification of LC3-positive phagosomes at 1 h after infection. Data are presented as the mean ± SEM of values from three independent experiments. ***p<0.001. (C) LC3-I and LC3-II levels in WT and <i>Vamp8</i>^-/- BMM infected for 1 h with opsonized WT or Δ<i>gp63 L</i>. <i>major</i> NIH S promastigotes or treated with 10 μM rapamycin were assessed by immunoblot analysis. LC3-II band intensities were measured by spot densitometry, normalized to the β-actin loading control and compared to the uninfected, untreated control (Ctl) cells.</p

<i>L</i>. <i>major</i> GP63 inhibits gp91^<i>phox</i> recruitment to the phagosome.

<p>(A) Confocal microscopy images of C57BL/6 peritoneal exudate macrophages (PEM) fed zymosan particles or infected with opsonized WT, Δ<i>gp63</i>, or Δ<i>gp63</i>+<i>gp63 L</i>. <i>major</i> NIH S promastigotes for 15 min. gp91^<i>phox</i> is in green; nuclei are in blue. Asterisks indicate phagosomes containing zymosan particles, white arrows indicate parasite nuclei, and red-filled arrowheads point to gp91^<i>phox</i> recruitment. Scale bar, 5 μM. (B) Quantification of gp91^<i>phox</i>-positive phagosomes at 15 min after infection. Data are presented as the mean ± SEM of values from three independent experiments. *p<0.05, **p<0.01. (C) gp91^<i>phox</i> levels in lysates from BALB/c BMM infected with opsonized WT, Δ<i>gp63</i> or Δ<i>gp63</i>+<i>gp63 L</i>. <i>major</i> NIH S promastigotes.</p

Infection with <i>L</i>. <i>major</i> promotes LC3 conversion and p62 expression.

<p>(A) Kinetics of LC3-I to LC3-II conversion induced by opsonized <i>L</i>. <i>major</i> NIH S promastigotes in BALB/c BMM was assessed by immunoblot analysis. (B) LC3-I and LC3-II levels in BALB/c BMM infected for 1 h with opsonized WT, Δ<i>gp63</i> or Δ<i>gp63</i>+gp63 <i>L</i>. <i>major</i> NIH S promastigotes was assessed by immunoblot analysis. (A, B) LC3-II band intensities were measured by spot densitometry, normalized to the β-actin loading control and compared to the uninfected control (Ctl) cells. (C) Kinetics of p62 accumulation in BALB/c BMM infected with opsonized WT or Δ<i>gp63 L</i>. <i>major</i> NIH S promastigotes was assessed by immunoblot analysis.</p

Down-modulation of VAMP3 and VAMP8 by <i>L</i>. <i>mexicana</i>.

<p>BMM were infected with serum-opsonized stationary phase <i>L</i>. <i>mexicana</i> (WT and <i>Δcpb</i>) promastigotes for 2 h. VAMP3 (<b>A</b>) and VAMP8 (<b>B</b>) levels (green) were then visualized by confocal microscopy. Macrophage and parasite nuclei are shown in blue (DAPI). Internalized parasites are denoted by white arrowheads. In <b>(C)</b>, VAMP3 and VAMP8 levels in total cell extracts were assessed by Western blot analysis. Each immunofluorescence assay was done on 300 phagosomes on triplicate coverslips in two independent experiments and Western blot analyses were performed twice in three independent experiments. VAMP3 and VAMP8/β-actin ratios were determined by densitometry. Original magnification X63.</p

<i>L</i>. <i>major</i> GP63 prevents LC3 recruitment to phagosomes.

<p>(A, C) Confocal microscopy images of BALB/c BMM infected for 1 h with opsonized WT, Δ<i>gp63</i>, or Δ<i>gp63</i>+<i>gp63 L</i>. <i>major</i> NIH S promastigotes. LC3 (A) and Beclin (C) are in green; nuclei are in blue. White arrows indicate parasite nuclei; red-filled arrowheads point to LC3 recruitment (A). Scale bar, 5 μM. (B) Quantification of LC3-positive phagosomes at 1 h after infection. Data are presented as the mean ± standard error of the mean (SEM) of values from three independent experiments. **p<0.01.</p

Absence of VAMP8 does not affect intracellular survival of <i>L</i>. <i>major</i> in macrophages.

<p>(A) Quantification of opsonized <i>L</i>. <i>major</i> GLC94 promastigotes internalization and replication in WT or <i>Vamp8</i>^-/- C57BL/6 x 129 BMM, assessed by Giemsa staining at 1, 24, 48, 72, and 96 h after infection. Data are presented as the mean ± SEM of values from three independent experiments. *p<0.05. (B) Images of Giemsa-stained WT or <i>Vamp8</i>^-/- BMM infected with opsonized <i>L</i>. <i>major</i> GLC94 for 2, 24, and 72h.</p

<i>L</i>. <i>major</i> GP63 cleaves VAMP8 and prevents its recruitment to phagosomes.

<p>(A) VAMP8 cleavage in C57BL/6 x 129 BMM infected for 2 h with opsonized WT, Δ<i>gp63</i> or Δ<i>gp63</i>+<i>gp63 L</i>. <i>major</i> 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, Δ<i>gp63</i>, or Δ<i>gp63</i>+<i>gp63 L</i>. <i>major</i> 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.</p

Expression of GP63 and LPG is impaired in the absence of CPB.

<p><b>(A)</b> Stationary phase promastigotes were lysed and total cell extracts were analysed by Western blotting and zymography for GP63 levels and activity and for LPG levels. Aldolase was used as a loading control. GP63 and LPG/aldolase ratios were determined by densitometry. <b>(B)</b> Promastigote total RNA was extracted and reverse transcription followed by PCR was performed to assess mRNA levels for <i>L</i>. <i>mexicana GP63-C1</i>, <i>LPG2</i>, and α-tubulin, and <i>L</i>. <i>major GP63-1</i>. Similar results were obtained in three independent experiments.</p

GP63 enables <i>L</i>. <i>mexicana</i> Δ<i>cpb</i> to infect macrophages and induce large PVs.

<p>BMM were infected with stationary phase serum-opsonized <i>L</i>. <i>mexicana</i> (WT, Δ<i>cpb</i>, Δ<i>cpb</i>+<i>cpb</i> and Δ<i>cpb</i>+<i>GP63</i>) promastigotes for 2 h, 24 h, 48 h and 72 h. Macrophages were stained with the HEMA 3 kit. Representative pictures from each condition are shown <b>(A)</b> Parasites were counted in 300 macrophages on triplicate coverslips <b>(B).</b> Macrophages were stained with the LAMP1 antibody and vacuole sizes were measured with the ZEN 2012 software <b>(C)</b>. Parasitemia and vacuole size was determined on 300 phagosomes in triplicate in three independent experiments. *p<0.0001.</p