Listeriolysin O promotes the intravacuolar growth of Listeria monocytogenes in epithelial cells

Upon entry into host cells, Listeria monocytogenes (Lm) was described to escape rapidly from internalisation vacuoles and proliferate only after gaining access to the cytosol. Vacuole escape depends upon three secreted virulence factors: the pore-forming toxin listeriolysin O (LLO) and two phospholipases. To quantify the dynamics of vacuolar escape, we used FAST fluorescent tags to monitor bacterial secretion into enclosed compartments. By tracking fluorescently-labelled vacuoles, we quantified the heterogeneity of 20 Lm residence time in primary vacuoles formed in epithelial LoVo cells. Although half of the bacterial population escaped from vacuoles within 13 minutes after internalisation, a fraction of it remained entrapped several hours in Long Residence Vacuoles (LRV), for both wild type and LLO-deficient strains. Unexpectedly, Lm replicated inside LRVs at a rate similar to that in the cytosol. LRVs were decorated with LLO-FAST and LLO was necessary for bacterial proliferation in these compartments, suggesting that 25 permeation of vacuolar membranes sustained growth. LRVs displayed similarities with the spacious Listeria-containing phagosomes described in macrophages, and could constitute an alternative replication niche for Lm in epithelial cells