Abstract
Real-time imaging of bacterial virulence factor dynamics is hampered by the limited number of fluorescent tools suitable for tagging secreted effectors. Here, we demonstrated that the fluorogenic reporter FAST could be used to tag secreted proteins, and we implemented it to monitor infection dynamics in epithelial cells exposed to the human pathogen Listeria monocytogenes (Lm). By tracking individual FAST-labelled vacuoles after Lm internalisation into cells, we unveiled the heterogeneity of residence time inside entry vacuoles. Although half of the bacterial population escaped within 13 minutes after entry, 12% of bacteria remained entrapped over an hour inside long term vacuoles, and sometimes much longer, regardless of the secretion of the pore-forming toxin listeriolysin O (LLO). We imaged LLO-FAST in these long-term vacuoles, and showed that LLO enabled Lm to proliferate inside these compartments, reminiscent of what had been previously observed for Spacious Listeria-containing phagosomes (SLAPs). Unexpectedly, inside epithelial SLAP-like vacuoles (eSLAPs), Lm proliferated as fast as in the host cytosol. eSLAPs thus constitute an alternative replication niche in epithelial cells that might promote the colonization of host tissues.
Highlights
Bacterial pathogens harness distinct colonization strategies to take advantage of their host resources
In order to measure the heterogeneity of Listeria monocytogenes (Lm) residence time in entry vacuoles and to assess the role played by listeriolysin O (LLO) in the dynamics of bacterial escape from these compartments, we developed live imaging assays allowing an accurate measurement of the time elapsed between the moment when individual bacteria were internalised into cells and the moment when the integrity of the vacuole membrane was disrupted
Fluorescent tagging with FAST of proteins secreted by Listeria monocytogenes
Summary
Bacterial pathogens harness distinct colonization strategies to take advantage of their host resources. Bacteria are entrapped inside primary vacuoles from where they can follow two distinct routes: either subverting endomembrane compartments, or leaving them. For instance Chlamydia trachomatis, Brucella abortus or Legionella pneumophila perturb the maturation and rearrange the properties of vacuoles, thereby creating a compartment prone to their replication [1]. Others, such as Shigella flexneri or Listeria monocytogenes, typically do not grow inside endomembrane compartments, but rather escape from entry vacuoles and gain access to the host cell cytoplasm, where they can replicate as well as exploit the host actin cytoskeleton for intracellular motility and cell-to-cell spread [2]
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