PT  - JOURNAL ARTICLE
AU  - Caroline Peron-Cane
AU  - José-Carlos Fernandez
AU  - Julien Leblanc
AU  - Laure Wingertsmann
AU  - Arnaud Gautier
AU  - Nicolas Desprat
AU  - Alice Lebreton
TI  - Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of <em>Listeria monocytogenes</em> in epithelial cells
AID  - 10.1101/2019.12.23.886689
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2019.12.23.886689
4099  - http://biorxiv.org/content/early/2020/09/18/2019.12.23.886689.short
4100  - http://biorxiv.org/content/early/2020/09/18/2019.12.23.886689.full
AB  - 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.Author summary Bacterial pathogens secrete virulence factors to subvert their hosts; however, monitoring bacterial secretion in real-time remains challenging. Here, we developed a convenient method that enabled fluorescent imaging of secreted proteins in live microscopy, and applied it to the human pathogen Listeria monocytogenes. Listeria has been described to invade cells and proliferate in their cytosol; it is first internalized inside vacuoles, from where it escapes thanks to the secretion of virulence factors that disrupt membranes. Our work revealed the existence, in human epithelial cells, of a population of Listeria that failed to escape vacuoles but instead multiplied efficiently therein, despite -and in fact, thanks to— the active secretion of a toxin that permeates membranes. This intravacuolar niche may provide Listeria with an alternative strategy to colonize its host.Competing Interest StatementThe authors declare the following competing financial interest: AG is co-founder and holds equity in Twinkle Bioscience/The Twinkle Factory, a company commercializing the FAST technology. FAST was patented by AG and L. Jullien (Patent Publication# WO/2016/001437, International Application# PCT/EP2015/065267).