Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells

Caroline Peron Cane (1, 2) , José Carlos Fernandez (1) , Julien Leblanc (1) , Laure Wingertsmann (1) , Arnaud Gautier (3, 4) , Nicolas Desprat (5, 1, 6) , Alice Lebreton (1)

1 IBENS - Institut de biologie de l'ENS Paris
2 LPENS - Laboratoire de physique de l'ENS - ENS Paris
3 LBM UMR 7203 - Laboratoire des biomolécules
4 IUF - Institut universitaire de France
5 UPD7 - Université Paris Diderot - Paris 7
6 ABCD : Biophysique des Biomolécules

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.

Mots clés: Virulence factors; Listeria monocytogenes; Bacterial secretion; Live cell imaging; Intravacuolar life; Pore-forming toxin; Host-pathogen interactions

Domaines: Bactériologie; Biologie moléculaire; Biophysique; Organisation et fonctions cellulaires [q-bio.SC]

Caroline Peron Cane, José Carlos Fernandez, Julien Leblanc, Laure Wingertsmann, Arnaud Gautier, et al.. Fluorescent secreted bacterial effectors reveal active intravacuolar proliferation of Listeria monocytogenes in epithelial cells. PLoS Pathogens, 2020, 16 (10), pp.e1009001. ⟨10.1371/journal.ppat.1009001⟩. ⟨hal-02424854v2⟩