Figure 6.

Listeria monocytogenes MVs inhibit autophagy induced by pore-forming toxin (VCC) associated with outer membrane vesicles (OMVs) of V. cholerae. HEK293-GFP-LC3 cells were incubated simultaneously with 250 μg MVs from the L. monocytogenes EGDe strain and 200 μg of V. cholerae V:5/04 OMVs (autophagy inducer) for 6 h (A–D) or 2.4 nM of purified V. cholerae cytolysin (VCC) for 5 h (E–H). Confocal images show (A,E) mock-treated (PBS) control cells, (B) cells treated with only V. cholerae V:5/04 OMVs and (C) cells treated with V. cholerae V:5/04 OMVs and EGDe MVs, (F) cells treated with only VCC and (G) cells treated with VCC and EGDe MVs. (I) and (J) represent magnified areas of the white squares in (B) and (C), respectively; (K) and (L) represent magnified areas of the white squares in (F) and (G), respectively. Scale bar: 10 μm. Green dots represent GFP-LC3 puncta, indicated with white arrows. The results represent at least three independent experiments. (D,H) Host cell lysates were analyzed for conversion of GFP-LC3-I (cytosolic) to GFP-LC3-II (membrane-conjugated) form by immunoblot using monoclonal GFP antiserum (upper panels). The membrane was reprobed using anti-α-actin antibody as an internal control. (D) Immunoblots show (a) mock-treated cells, (b) cells treated with only V. cholerae OMVs, and (c) cells treated with MVs from both bacteria. (H) Immunoblots show (a) mock-treated cells, (b) cells treated with only VCC, and (c) cells treated with VCC and EGDe MVs. The ratio of GFP-LC3-II to α-actin was quantified from three independent experiments, normalized and presented in arbitrary units (lower panels). ^**P < 0.01, ^*P < 0.05, Student's t-test.