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

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.

Pathogenicity and virulence of Listeria monocytogenes: A trip from environmental to medical microbiology

“…SLAPs are nonacidic and non-degradative phagosomes generated in an autophagy dependent manner, which maintain a sub-population of intracellular L. monocytogenes producing low amounts of LLO. More recently, live fluorescent microscopy of Listeria infection of epithelial cells revealed that a subset of bacteria remains within long-term vacuoles, where they can proliferate as quickly as cytosolic ones [ 238 ]. Cytoplasmic L. monocytogenes has also been shown to switch from an active motile lifestyle to a stage of persistence within vacuoles in epithelial and trophoblast cells during several days of infection.…”

Engineering of a fluorescent chemogenetic reporter with tunable color for advanced live-cell imaging

“…Prototypical FAST is a small protein tag (14 kDa) with reduced genetic footprint, which was originally designed to fluoresce instantaneously upon non-covalent binding of fluorogenic 4-hydroxybenzylidene rhodanine (HBR) derivatives that are inherently non-fluorescent unless bound to FAST 13 . Recent studies showed that FAST enabled (i) to circumvent some limitations of conventional fluorescent proteins—allowing for instance the imaging of proteins in strict anaerobic organisms 14 , 15 or the visualization of protein secretion by bacteria in real time 16 , 17 —and (ii) to bioengineer functional biosensors for monitoring intracellular analytes 18 , and observing protein–protein interactions with high spatial and temporal resolution 19 . Capable of binding various membrane permeant and membrane-impermeant HBR derivatives 13 , 20 , 21 , prototypical FAST exhibited great potential for engineering a multifunctional fluorescent chemogenetic reporter with tunable color through chromophore modulation.…”

Inhibition of the master regulator of Listeria monocytogenes virulence enables bacterial clearance from spacious replication vacuoles in infected macrophages

“…As these vacuoles shared features of SLAPs [ 18 ], but supported extensive intravacuolar L . monocytogenes replication as reported for eSLAPs [ 22 ], we named them replication-permissive SLAPs (rSLAPs). At the time of high intra-vacuolar L .…”

“…In epithelial cells, epithelial SLAP-like vacuoles (eSLAPs) were recently reported. eSLAPs share some features with SLAPs such as decoration with LC3, LAMP1, as well as neutral pH [ 22 ]. However, eSLAPs support rapid intravacuolar L .…”

“…However, eSLAPs support rapid intravacuolar L . monocytogenes replication (generation time 102 min), similar to bacteria residing in the cytoplasm [ 22 ]. These entry-vacuole-derived eSLAPs are distinct from single-membrane Listeria -containing vacuoles (LisCVs) that were observed in long-term in vitro cultures of human trophoblast cells and hepatocytes [ 23 ].…”