Figure 4.

Listeriolysin O localization and activity inside the MVs. (A) Immunoblot detection of listeriolysin O (LLO) in density gradient fractions of isolated and purified MVs. Fractions are numbered from left to right (1–23) according to increasing density. Fractions 1-5 containing soluble LLO do not show MVs under TEM; fractions 15-20 contain MVs as shown in Figure 1C. Polyclonal anti-LLO antiserum was used to detect LLO at 56 kDa using SDS-PAGE and immunoblot analysis of all the fractions. (B) LLO is tightly associated with MV structure. Dissociation assay was performed using 1 M NaCl, 0.1 M Na₂CO₃, 0.8 M urea or 1% SDS. After incubation, MVs were ultracentrifuged, and supernatant (S) and pellet (P) fractions were analyzed by immunoblot using a polyclonal anti-LLO antiserum. (C) MV-associated LLO shows no hemolytic activity unless exposed to reducing conditions. Hemolytic activity of MVs from EGDe (wt) and the Δhly strain in comparison with purified LLO containing DTT. Goat erythrocytes (10%) were incubated either with LLO (100 ng or 50 ng) or with MVs (containing 1.15 μg LLO or 230 ng LLO) for 3 h at 37°C. Where indicated, MVs were pre-incubated with a reducing agent, 2 mM DTT, for 20 min at 25°C. Hemolytic activity of 100 ng LLO was normalized up to 100%; 2 mM DTT was used as a negative control. The mean with SD is shown. Results represent three independent experiments. ^**P < 0.01, ^***P < 0.001, Student's t- test.