To directly test whether membrane damage induced by LLO was sufficient for autophagy activation during the early stages of infection, we incubated macrophages with liposomes containing either active or heat-killed LLO (HK-LLO) in the presence of Texas Red dextran as a fluid-phase marker [31, 32] . As previously reported, LLO-containing liposomes were phagocytosed, their contents released and the dextran delivered to the cytosol. Of the .300 liposomes counted at each time point over three independent experiments, we found that liposomes encapsulating wild-type LLO had a peak of LC3 colocalization (8%) at 25 minutes post-infection (Fig. 4A,B) . The liposomes encapsulating HK-LLO showed less than 2% colocalization with LC3 at the same time point (Fig. 4B ). Because only ,10% of phagosomes harboring LLO-loaded liposomes are competent to rupture (as measured by dye release; KD Lee, personal communication), 8% was not statistically significantly different from this 10% expectation, while 2% was statistically significantly different from both the expected (10%) and observed (8%) values (p..99 by z-test, using a binomial distribution). These data are consistent with a model in which LLO-mediated membrane damage is sufficient to induce LC3 colocalization in the absence of bacteria.