An alternative replication niche for Listeria monocytogenes revealed by fluorescent tagging of bacterial secreted effectors

Tracking the dynamics of secreted virulence factors in real time is impaired by the paucity of appropriate fluorescent tools. Here, we took advantage of the fluorogenic reporter FAST to tag secreted proteins and monitor infection dynamics among a population of epithelial cells infected by Listeria monocytogenes (Lm). Thereby, we unveiled the heterogeneity of Lm residence time in internalisation vacuoles. Although half of the bacterial population escaped rapidly, a significant fraction remained entrapped several hours in Long Residence Vacuoles (LRVs), independently of the secretion of the pore-forming toxin listeriolysin O (LLO). Unexpectedly, LLO enabled Lm to proliferate inside these compartments as fast as in the cytosol. LRVs display similarities with the spacious Listeria-containing phagosomes described in macrophages, and could constitute an alternative replication niche for Lm in epithelial cells. Listeria intravacuolar replication


Introduction
Bacterial pathogens harness distinct colonization strategies to take advantage of their host resources. While some remain extracellular, others adopt an intracellular lifestyle. Internalisation into host cells provides invasive bacteria with multiple abilities, such as that of crossing the organism barriers, escaping humoral immune surveillance, or disseminating throughout the organism as cargo of circulating cells. After internalisation, bacteria are entrapped inside primary vacuoles from where they can follow two distinct routes: either subverting endomembrane compartments, or leaving them. For instance, Mycobacterium tuberculosis, Chlamydia trachomatis, Brucella abortus, Coxiella burnetii, Legionella pneumophila perturb the maturation and rearrange the properties of vacuoles, thereby creating a compartment prone to their replication (Di Russo Case and Samuel, 2016;reviewed in Salcedo and Holden, 2005). 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 (reviewed in Gouin et al., 2005).
The foodborne pathogen Listeria monocytogenes (hereafter, Lm) is the causative agent of listeriosis, and has emerged as a model facultative intracellular bacterium (reviewed in Cossart and Lebreton, 2014;Radoshevich and Cossart, 2017). This pathogen can cross the protective barriers of its host and colonize tissues and organs by promoting its internalisation into non-phagocytic cells. The classical scheme of Lm intracellular life cycle implies that, both in professional phagocytes and in epithelial cells, Lm rapidly escapes from entry vacuoles due to the combined action of a potent pore-forming toxin, listeriolysin O (LLO), and of two phospholipases C (PlcA and PlcB), before replicating in the cytosol (Pizarro-Cerda and . All three genes (hlyA that encodes LLO, plcA and plcB) are part of Lm LIPI-I virulence gene cluster and are transcriptionally induced by PrfA, the main regulator of Lm virulence gene, in intracellular bacteria (reviewed in las Heras et al., 2011;Lebreton and Cossart, 2017).
LLO is a cholesterol-dependent pore-forming toxin (reviewed in Nguyen et al., 2018), secreted by Lm via the general secretion system (Sec). LLO assembles into oligomers on biological membranes, forming arcs and pores of several tens of nm that disrupt membrane integrity (Köster et al., 2014;Ruan et al., 2016). Its activity is optimal at acidic pH representative of the acidification occurring during the maturation of phagosomes (pH = 4.9 to 6.7) (Beauregard et al., 1997;Schuerch et al., 2005), which has been proposed to facilitate the escape of bacteria from entry vacuoles while avoiding damages to the host plasma membranes at neutral pH.
Whereas LLO-deficient Lm cannot gain access to the host cytosol in many cell types, the activity of the phospholipases PlcA and PlcB and the influence of host factors render LLO dispensable for vacuole escape in several human epithelial cell lines (Burrack et al., 2009;Marquis et al., 1995). In phagocytes, it has been shown that bacteria secreting reduced amounts of LLO could remain entrapped in long-term compartments named Spacious Listeria-containing Phagosomes (SLAPs) and even replicate extremely slowly therein, with a doubling time in the range of 8 h (Birmingham et al., 2008).
The escape dynamics from the entry vacuole has been experimentally addressed using several distinct strategies. One of them consisted in using medium containing a membrane-impermeant fluorescent dye during infection (Beauregard et al., 1997;Myers et al., 2003). Upon encapsulation into the internalisation vacuoles together with invading bacteria, the fluorescent dye stained the intravacuolar space until it broke down.
Alternative strategies were based on the assessment of vacuole rupture events and bacterial access to the host cytosol using fluorescent sensors. For instance, galectin-3 has been shown to label membrane remnants of damaged vacuoles and thereby allow the spotting of vacuole lysis (Paz et al., 2010). Likewise, actin or the Cell-wall Binding Domain CBD (a domain from the Lm phage endolysin Ply118) are recruited to the bacterial surface only once Lm has escaped the vacuole (Henry et al., 2006;Pizarro-Cerda and Cossart, 2018).
Cytoplasmic FRET probes that are cleaved by a b-lactamase secreted by invasive bacteria have also been described as efficient reporters of vacuole rupture (Quereda et al., 2015;Ray et al., 2010). Even though these approaches yielded the order of magnitude of the time lapse between bacterial entry and vacuole escape in various cell types (between 15 min and 1 h), none of them has been used so far to determine the distribution of Lm residence time in entry vacuoles, which limits the interpretation of variations between conditions to that of average behaviours.
In order to measure the heterogeneity of Lm residence time in entry vacuoles and to assess the role played by 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 vacuole membrane was ruptured. We devised a strategy relying on the tagging of proteins secreted by bacteria with the FAST reporter system (Plamont et al., 2016).
FAST is a 14-kDa protein tag which displays fluorescence upon binding with a synthetic fluorogenic probe supplied in the medium. The fluorogen is membrane permeant, non-toxic, and has very little fluorescence by itself. The small size of FAST, its fast folding kinetics, the reversible binding of fluorogens together with good brightness and photostability made this system an ideal candidate for tagging secreted proteins and imaging them in real time.
Using live imaging of FAST, we quantified the distribution of Lm residence times in primary vacuoles in the LoVo intestinal epithelial cell line. We observed that a fraction of the population of entry vacuoles lasted for several hours and were reminiscent of SLAPs. However, in contrast with SLAPs, these Long Residence Vacuoles (LRVs) were obtained in cells infected with wild type (WT) Lm as well as with a hlyA deletion strain.
Furthermore, secretion of LLO inside LRVs allowed Lm to proliferate actively in these compartments, suggesting that besides its role in vacuole escape, LLO could contribute to set up an intravacuolar niche prone to Lm replication in epithelial cells.

Fluorescent tagging with FAST of proteins secreted by Listeria monocytogenes
With the aim of detecting proteins that would be secreted by intracellular bacteria into their host cells in live-cell microscopy experiments, we explored the possibilities offered by the FAST reporter system for the fluorescent tagging of Lm secreted bacterial effectors. A set of integrative plasmids harbouring gene fusions under control of the PHYPER promoter were designed ( Figure 1A) and introduced in the genome of Lm strain LL195. These plasmids drove the constitutive production of either FAST or eGFP, either for intrabacterial localisation, or fused in their N-terminus to the secretion signal peptide (SP) of listeriolysin O (LLO) (SP-FAST and SP-eGFP constructs), or to full-length LLO (LLO-FAST, LLO-eGFP and untagged LLO constructs), a classical Sec substrate. A Myc tag in their C-terminus of all constructs allowed detection by immunoblotting.
Protein production and secretion by each one of these seven strains was assessed by in-gel colloidal Coomassie staining and immunoblotting against the Myc tag, on bacterial total extracts and culture supernatant fractions from overnight-grown cultures in BHI ( Figure S1). All transgenes were efficiently expressed by Lm, even though in varying amounts. As expected, constructs harbouring either the LLO SP, or full-length LLO, were recovered in bacterial culture supernatants, indicating that the SP of LLO promoted Sec-dependent export of FAST or FAST-tagged proteins, as well as eGFP-fusion proteins albeit to a lesser extent ( Figure S1C, D).
Constructs devoid of signal peptides were not detected in supernatant fractions, arguing against the release of proteins into the medium due to bacterial lysis. FAST-tagged Sec substrates can thus efficiently undergo secretion trough the general secretion pathway.
To assess whether the FAST reporter system remained fluorescent after secretion, we quantified the fluorescence signals in the culture medium of overnight-grown bacterial cultures in iLSM ( Figure 1B). In presence of 5 μM of HBR-3,5DM, fluorescence was detected in the culture supernatant of strains secreting SP-FAST or LLO-FAST. Fluorescence intensities in the culture medium of strains producing non-secreted FAST or eGFP did not significantly differ from that of the strain producing untagged LLO, indicating that the release of fluorescent proteins in the culture medium was not due to bacterial lysis, and that FAST-labelled proteins retained their fluorescent properties after undergoing secretion trough Sec. By calibrating fluorescence measurements with a standard curve of known FAST:HBR-3,5DM concentrations diluted in the same minimal medium, we estimated the secreted concentration of tagged proteins; that of SP-FAST was 325±55 nM, and that of LLO-FAST around 28±6 nM.
Diverse attempts by others in Gram-negative bacteria (Dammeyer and Tinnefeld, 2012) and our own unpublished observations using tagged Lm virulence factors suggested that the Sec-dependent secretion and subsequent maturation of an eGFP tag into its active, fluorescent fold was inefficient.
Surprisingly, the secretion of SP-eGFP-but not that of LLO-eGFP-gave rise to fluorescent signals in culture supernatants, even though in a range 10-fold lower than that obtained for the secretion of SP-FAST ( Figure 1A).
A consistent proportion of eGFP undergoing Sec-dependent secretion was thus able to acquire its mature fold in bacterial culture medium, at least in iLSM, and when not fused to a bacterial effector.

Fluorescent tagging with FAST of Shigella effectors secreted through the type III secretion system
To evaluate the versatility of FAST as a reporter of bacterial secretion, we next asked if FAST was suitable for fluorescent tagging of effectors secreted through the syringe of the type III secretion system (T3SS) from a Gram-negative pathogen, Shigella flexneri (Sf) strain M90T. As model T3SS substrates, we tagged Cterminally with FAST-Myc the effectors OspF and IpaB ( Figure 1C), which are translocated upon adhesion of Sf to host cells (reviewed in Pinaud et al., 2018). Bacterial total extracts and culture supernatant fractions were recovered from overnight-grown cultures in M9 medium, with or without stimulation of type-III dependent secretion by addition of Congo red. By immunoblotting these fractions against the Myc epitope, we observed that tagged OspF and IpaB were secreted into the bacterial culture medium upon Congo red induction ( Figure   S2A). The secretion of both tagged effectors was constitutive when using a ∆ipaD mutant strain for which translocation lacks gating controls (Ménard et al., 1994) ( Figure S2B). We then assessed whether the fusion proteins secreted by the ∆ipaD strain had retained their fluorescent properties, by measuring fluorescence intensities in the supernatants of bacterial cultures grown overnight in M9 medium ( Figure 1D). Fluorescence levels were consistently higher with this constitutively secreting strain than the fluorescence leakage measured for the WT strain when the T3SS was not induced. The concentration of OspF-FAST by the ∆ipaD strain was estimated to be 3.8 ± 0.3 nM, that of IpaB-FAST of 9.4 ± 1.7 nM. Like Sec substrates, FAST-tagged T3SS substrates can thus be translocated and keep fluorescent properties after secretion.

FAST-tagging of secreted Listeria effectors for live fluorescence microscopy
We next investigated whether the FAST reporter system was suited for intracellular detection in real-time microscopy of proteins secreted during infection. To this end, we monitored FAST signals in LoVo cells infected with Lm producing SP-FAST by confocal spinning disk microscopy over an infection time course

Residence time of Listeria monocytogenes in internalisation vacuoles
When FAST-tagged proteins were secreted into the large volume of the host cell cytoplasm, fluorescent signals were diluted and therefore only became visible after several hours of infection, once secreted FAST had accumulated sufficiently to be significantly discriminated from non-specific signals. Meanwhile, we reasoned that if Lm was confined inside micron-sized internalisation vacuoles, the higher concentration of secreted FAST molecules in a reduced volume would allow their detection and tracking until the rupture of vacuole membranes, thereby providing an accurate measurement of individual vacuole lifetimes ( Figure 3A).
Indeed, we observed that secreted FAST signals were enhanced in compartments that co-localized with mCherry-expressing bacteria within minutes after bacterial adhesion to cells, until these signals suddenly dropped when vacuoles ruptured ( Figure 3B, Movie S2).
We used SP-FAST secretion to track intravacuolar fluorescent signals, and compare the residence time of WT or ∆hlyA Lm strains inside internalisation vacuoles formed in LoVo cells ( Figure 3C-D). The hlyA deletion strain used in this experiment was generated by in-frame allelic replacement of the hlyA open reading frame with SP-FAST (∆hlyA::SP-FAST, Figure S3A). The median value for the residence time of the WT strain was of 12.7±0.7 min ( Figure 3D). When using the ∆hlyA::SP-FAST strain, the median residence time was significantly longer (21.1 ± 1.4 min) but remained of the same order of magnitude as for a strain producing LLO, confirming previous observations that Lm gained efficient access to the cytoplasm independently of LLO in epithelial cells (Burrack et al., 2009;Marquis et al., 1995). Unexpectedly, a large proportion of the entry vacuoles lasted for more than one hour (12.0 % for the WT strain; 14.8 % for the ∆hlyA mutant), and a consistent number of intact vacuoles was still observed 3 h p.i. (4.6 % for the WT strain; 6.2 % for the ∆hlyA mutant) ( Figure 3C). The fact that the WT strain remained entrapped in Long Residence Vacuoles (LRVs) in proportions nearly identical to that of the ∆hlyA strain could either suggest that a sub-population of WT Lm failed to escape primary vacuoles in spite of LLO secretion, or that LLO was not produced by this subpopulation of intravacuolar bacteria. To discriminate between these two hypotheses, we investigated whether LLO fused to a FAST tag was detected in vacuoles out of which Lm had failed to escape.

Long-term residence and rapid replication of Listeria inside LLO-decorated vacuoles
To examine whether LLO was produced and secreted by bacteria that remained entrapped in LRVs, we engineered a Lm strain where LLO was C-terminally fused with FAST at the endogenous hlyA locus ( Figure S3A). In this strain, the fluorescence of FAST reported not only for LLO secretion and localisation, but also for hlyA expression under its natural promoter. In order to be relevant for monitoring the dynamics of Lm intracellular infection, the 15-kDa FAST-Myc tag should not interfere with the function of the protein it reports for. We controlled that the haemolytic properties of the strain expressing hlyA-FAST did not differ from that of the WT strain ( Figure S3B); the production, secretion and activity as a cytolysin of LLO are thus quantitatively and qualitatively preserved after C-terminal fusion with FAST.
The strain producing the LLO-FAST fusion also constitutively expressed mCherry, which allowed us to segment and track bacteria in 3D during infection. When imaging mCherry-labelled bacteria and LLO-FAST from 2 h post-infection (p.i.) in LoVo cells, we observed that Lm could remain entrapped inside LRVs for 9 h or more before the enclosed structure of LLO-labelled membranes eventually disrupted and bacteria dispersed into the cytosol ( Figure 4A, Movie S3). Strikingly, the volume occupied by the mCherry signal increased over time, revealing that not only Lm inhabited LRVs for a long time, but that it was multiplying inside these compartments. The ability of Lm to grow inside LLO-FAST-labelled vacuoles was observed for both the LL195 genetic background (Lm lineage I, ST1) and the European Lm reference strain EGD-e (lineage II, ST9) ( Figure S4A, Movie S4), indicating that this property was not specific to the hypervirulent clone LL195.
Likewise, the proliferation of Lm inside LRVs was observed in Caco-2 cells, suggesting that LoVo cells were not the only epithelial niche allowing Lm to replicate inside endomembrane compartments ( Figure S4B).
By tracking vacuoles and measuring their volumes over time, we determined the growth rate of WT Lm in LRVs. Intravacuolar growth was exponential, with a rate similar to that of free bacteria in the cytosol ( Figure   4B, Figure S5A). The measured doubling time (90 min in this experimental setup; or down to 1 h when the intensity and frequency of illumination were reduced as in Figure 2D) was consistently shorter than that previously described in SLAPS, which was in the range of 8 h (Birmingham et al., 2008).

Role of listeriolysin O in the long-term intravacuolar residence and replication of Listeria
Our observations suggested that even when LRVs were permeated by LLO, their integrity was maintained and they allowed intravacuolar replication without rupturing. To further investigate whether LLO influenced Lm residence in LRVs, we took advantage of the LLO-FAST reporter strain in order to assess the variability in LLO abundance in these compartments. LLO-FAST signals measured in LRVs displayed a broad range of dynamics, indicating that LRV formation and preservation was independent of the amounts of secreted LLO ( Figure S5B). In some LRVs, LLO-FAST accumulated linearly over time, while others displayed large-scale fluctuations in signals. Some LRVs yielded a strong signal while others displayed low levels of decoration by LLO. The lifetime of LRVs was correlated with neither the average concentration of LLO ( Figure S5C) nor its maximal level ( Figure S5D), suggesting that LLO concentration poorly influenced the probability of Lm escape from these structures.
Consistently with the absence of correlation between LLO abundance and bacterial escape from LRVs, not only WT and ∆hlyA Lm could reside in long-term vacuoles ( Figure 3C, Figure 4C, Figure S3C), but LRVs were even observed when using a prfA* mutant strain. This strain carries a prfA allele encoding a PrfA variant with a G145S substitution that has been previously described to be constitutively active, and to lead to the strong overexpression of PrfA-dependent virulence genes, including that of hlyA (Ripio et al., 1997).
Accordingly, the in-vitro haemolytic titre of the prfA strain we used was fifty-fold higher than that of the WT strain, indicative of LLO hyperproduction ( Figure S3B). Highly elevated levels of secreted LLO thus did not impede the ability of Lm to reside and multiply inside LRVs for several hours. This feature is contrasting with that of SLAPs previously described to occur in phagocytes only when the expression of hlyA was moderate (Birmingham et al., 2008). Nevertheless, we observed by live-cell imaging that the very high levels of LLO secretion of the prfA* strain hastened Lm escape from LRVs to a certain extent ( Figure 4C, Figure S3C). In agreement with this result, the proportion of bacteria that replicated inside LRVs was lower when cells were infected with the prfA* strain than with the WT strain ( Figure 4D), confirming that a major excess of LLO had a destabilising effect on the integrity of LRVs.
Although LLO was not necessary for LRV formation or Lm vacuole escape, we observed that the ∆hlyA Lm strain was unable to proliferate inside LRVs ( Figure 4C, Figure S3C). Similar to SLAPs, LRVs thus required that bacteria secreted LLO to allow intravacuolar growth. Whereas the secretion of LLO was required for proliferation in LRVs, its quantity did not influence intravacuolar bacterial growth, since the prfA* strain replicated at a similar rate as the WT strain in LRVs ( Figure 4C). Consistently, the growth rate of LLO-FASTsecreting bacteria in LRVs was correlated with neither the average concentration ( Figure S5E) nor the maximal level of LLO secretion ( Figure S5F).

Origin and properties of Listeria long residence vacuoles in epithelial cells
The LRVs in which Lm replicated (Figure 4, Movies S3-4) likely originated from internalisation vacuoles from which bacteria had failed to escape ( Figure 3C), unless they derived from secondary vacuoles produced by cell-to-cell spread, or by autophagy vacuoles where bacteria would have been entrapped after a first exposure to the cytoplasm. To assess whether the LRVs where Lm proliferated resulted from primary vacuoles, we monitored the intravacuolar stages of mCherry-expressing bacteria in LoVo cells transfected by the YFP-CBD fusion protein reporter (Henry et al., 2006). This reporter has been previously described to specifically label the surface of bacteria that have once been exposed to the host cytosol, because the cell wall-binding domain (CBD) from the Lm phage endolysin Ply118 binds the peptidoglycan of Lm with high affinity. Bacteria that replicated within LRVs remained unlabelled with YFP-CBD until the vacuole ruptured and bacteria dispersed throughout the cell ( Figure 5A, Movie S5), indicating that they had not been in prior contact with the host cytosol. This result ruled out the possibility that bacteria became entrapped into secondary vacuoles by canonical autophagy or cell-to-cell spread after a first exposure to the host cell cytosol, and thereby confirmed that LRVs where Lm replicated originated from internalisation vacuoles.
Because the replication compartments we observed were reminiscent of SLAPs, we hypothesized that they could originate from a process analogous to LC3-associated phagocytosis (LAP), except it would occur in epithelial cells rather than in phagocytes. We thus endeavoured to further characterize this intravacuolar replication niche, and analyse whether it had typical features of endosomal, lysosomal and/or noncanonical autophagy-derived compartments. By immunofluorescence staining of LoVo cells infected with mCherryexpressing Lm for 3 hours, we observed that the vacuoles containing several bacteria were negative for the early endosomal marker Rab5, while they were positive for the late endosomal marker Rab7, the lysosomal marker LAMP1, as well as LC3 ( Figure 5B). These are typical markers of SLAPs, suggesting that, similar to what occurs in phagocytes, LC3 is lipidated and the noncanonical autophagy machinery recruited to the entry vacuole in epithelial cells. Also, as in SLAPs the pH inside LRVs remained neutral, as revealed by their absence of staining when using the acidophilic fluorescent probe LysoTracker Deep Red ( Figure 5B).
Altogether, we conclude that epithelial LRVs display molecular characteristics highly reminiscent of SLAPs, even though they allow a faster replication of Lm, and their maturation and rupture is less sensitive to the concentration of secreted LLO than the compartments observed in phagocytes.

Discussion
Exploring the dynamics of secreted virulence factors at the (sub-)cellular scale constitutes one of the main challenges for real-time microscopy of infectious processes. Here, we bring evidence that FAST offers a versatile, convenient opportunity for tackling this challenge. We took advantage of this system to measure the lifetime of Lm internalisation vacuoles, and to monitor the endomembrane localisation of the secreted Lm virulence factor LLO in live cells. As a result, we uncovered a new replicative niche for Lm in epithelial cells.

Real-time imaging of LLO during infection
On fixed samples, observing the localisation of LLO in infected cells has often constituted a hurdle, due to the poor quality of the labelling allowed by existing anti-LLO antibodies in immunofluorescence assays (e.g. Henry et al., 2006). LLO localisation at vacuole membranes, or more recently in bacterial-derived membrane vesicles, was previously observed by electron microscopy using immunogold labelling (Coelho et al., 2018;Quinn et al., 1993). However, the precise dynamics of infectious processes cannot accurately be caught by fixed-cell studies. Besides, the high spatial resolution gained by electron microscopy compromises the observation of events at a cellular scale. As a complementary approach, LLO-eGFP fusions that were ectopically-expressed in host cells have enabled live imaging, yielding precious insight into the dynamics of LLO localisation at membranes and its turnover (Chen et al., 2018). Nevertheless, ectopic expression by host cells cannot mimic the concentrations, location, and insertion into membranes from the inside of the vacuole obtained with bacterial secretion. Moreover, host cell signalling pathways and membrane dynamics differ between non-infected and infected cells. Here, we report that (a) the FAST system can be used to tag LLO without loss of function, (b) the LLO-FAST fusion, expressed from its endogenous promoter, is secreted by Lm in infected cells, (c) the vacuoles it decorates can be imaged with accuracy, and (d) some of these vacuoles unexpectedly last for several hours.

FAST, a versatile fluorescent reporter of bacterial secretion
Beyond the live detection of LLO secreted by L. monocytogenes through the general Sec secretion system, FAST opens new perspectives for real-time imaging of bacterial proteins secreted by a broader range of bacterial models and secretion systems. For instance, we provide evidence that FAST-tagged effectors are also efficiently secreted through the T3SS of S. flexeri.
In recent years, several strategies have emerged for fluorescent labelling of Sec-or T3SS-dependent substrates (reviewed in O' Boyle et al., 2018). Tagging bacterial effectors with Split-GFP provides a possible solution that has been successfully applied for live detection of Salmonella T3SS-dependent effectors or Listeria Sec-dependent secreted substrates (Batan et al., 2018;Van Engelenburg and Palmer, 2010); however, the reconstitution process is slow compared with microbial growth, and requires the stable expression of GFP1-10 in recipient cells, which limits its application in most biological systems. Superfolder GFP (sfGFP) or its derivative rsFolder have been successfully used for labelling E. coli periplasmic proteins exported through the Sec pathway (Dammeyer and Tinnefeld, 2012;Dinh and Bernhardt, 2011;Khatib et al., 2016;Peters et al., 2011), but to our knowledge has not been applied yet for other bacterial systems or in the context of hostpathogen interactions. Other fluorescent tags such as FlAsH and phiLOV were successfully used for monitoring the secretion of Sf T3SS-dependent effectors (Enninga et al., 2005;Gawthorne et al., 2016).
Nevertheless, the toxicity in eukaryotic cells of the biarsenite dye used for FlAsH labelling and the rather modest brightness of phiLOV hamper their general use.
FAST compares with previously existing tools, while broadening the possible range of applications, due to (a) its ease of implementation (compared with Split-GFP); (b) its low toxicity (compared with FLASH); (c) its independence to oxygen allowing studies in anaerobes (Monmeyran et al., 2018;Streett et al., 2019) as well as (d) its rapid and reversible folding dynamics allowing transport through the T3SS (compared with GFPderived probes); (e) its reasonable brightness and fast maturation time (compared with PhiLOV). In addition, by selecting the appropriate fluorogen, FAST-labelled proteins can be imaged at different wavelengths between 540 and 600 nm by selecting the appropriate fluorogen (Li et al., 2017), thereby providing users with flexibility in the choice of other fluorescent reporters in co-localisation studies. Red-shifted fluorogens also limit the toxicity of certain wavelength for bacteria when performing long-term imaging, and membraneimpermeant fluorogens offer the possibility to discriminate between intracellular and extracellular proteins (Li et al., 2018), for instance when addressing the localisation of bacterial effectors that anchor to the bacterial cell wall or to membranes.
Hence, FAST expands the panel of fluorescent reporters for monitoring secreted virulence factors and offers a wealth of opportunities to accurately seize the spatiotemporal aspects of infectious mechanisms.

LRVs, an alternative replication niche for Listeria monocytogenes
We document that in LoVo and Caco-2 epithelial cells, a consistent proportion of Lm fails to escape from internalisation vacuoles, but instead replicates efficiently inside LRVs, which are positively labelled by LLO-FAST ( Figure 6). After several hours of intravacuolar residence and growth, the membrane of LRVs eventually rupture and bacteria resume a canonical cytosolic lifestyle.
The decoration of LRVs by LC3, Rab7 and LAMP1 as well as their neutral pH are reminiscent of the SLAPs previously described in phagocytes (Birmingham et al., 2008), and which derive from LAP (Mitchell et al., 2018). A process similar to LAP was also described to occur in epithelial cells during infection by Sf Whereas the LRVs that we observe in LoVo cells display similarities with SLAPs, they are notably distinct from LisCVs, an intravacuolar persistence niche of Lm recently described in human hepatocytes and trophoblast cells (Kortebi et al., 2017). Contrary to LRVs and SLAPs, LisCVs do not derive from primary vacuoles. Instead, they form late in the intracellular cycle of Lm by recapture of bacteria that have lost ActAdependent motility. Indeed, bacteria found in LisCVs are labelled with YFP-CBD, while the bacteria we observe in LRVs are not. ActA function is indifferent to the formation of LRVs, since they are also observed when using a prfA* strain, in which actA is constitutively expressed (Reniere et al., 2015). Moreover, whereas LRVs are lipidated by LC3, LisCVs are not. Last, Lm replicates in LRVs, whereas it adopts a viable, nonculturable state LisCVs and does not grow. Altogether, though occurring in epithelial cells, the features we describe for LRVs are consistent with compartments similar to SLAPs, and distinct from LisCVs. However, the replication of Lm inside LRVs is significantly faster than the 8 hours of doubling time reported in SLAPs (Birmingham et al., 2008), perhaps due to a lower bactericidal capacity of the epithelial niche compared with phagocytes. Membrane permeation by LLO might also attenuate the bactericidal properties of LRVs, and/or allow nutrient uptake through the permeated membrane, thereby promoting bacterial replication.
Together with LisCVs and SLAPs, LRVs enrich the palette of Lm intravacuolar lifestyles that can establish in various cells types. Apprehending the importance of LRVs in the context of in vivo infections prompts future investigation, as these compartments might provide shelter from cytosolic immune responses, or favour chronic forms of infections by dampening cell-to-cell spread within tissues. Deciphering the extent to which these intravacuolar niches influence the balance between bacterial fitness and host defences becomes critical to better appreciate long-term relationships between Lm and its host.

Bacterial strains, plasmids and culture conditions
The bacterial source strains used in this work were Escherichia coli NEB5a (New England Biolabs) for plasmid constructions, Rosetta(DE3)pLysS (Novagen) for recombinant protein production, the clinical isolate 20 μg/ml for Sf; 7 μg/ml for Lm or Ampicillin, 100 μg/ml), or Congo red (1 mg/ml) for activation of the Sf

T3SS.
In order to favour the expression of transgenes, the DNA coding sequence for FAST, fused with a Myctag, was codon-optimized for Lm or Sf using the online Optimizer application (http://genomes.urv.es/OPTIMIZER/) in guided random mode. The optimized sequences (named gfAL001 and gfAL002, respectively) were obtained as synthetic Gene Fragments (Eurofins genomics) and will be provided upon request. The Lm-optimized sequence additionally contained the 5'-untranslated (5'-UTR) of the hlyA gene, and the sequence encoding the signal peptide (SP) of LLO in its N-terminal part.
For plasmid constructions in the pAD vector derived from the pPL2 backbone (Balestrino et al., 2010;Lauer et al., 2002), gfAL001 was amplified with primers oAL543-4, the sequence of the Lm hlyA gene encoding LLO was amplified from the EGD-e genomic DNA with primers oAL549-50b, and the coding sequence for eGFP was amplified from pAD-cGFP (BUG2479) (Balestrino et al., 2010) with primers oAL543-7. The UTRhlyA-SP-FAST-Myc amplicon was inserted instead of UTRhlyA-eGFP into the EagI-SalI restriction sites of pAD-cGFP, thus generating pAD-SP-FAST, where FAST is under control of the PHYPER constitutive promoter ( Figure 1A). pAD-FAST, pAD-eGFP, pAD-SP-eGFP, pAD-LLO, pAD-LLO-FAST and pAD-LLO-eGFP, all containing the 5'-UTR of hlyA and a Myc tag, were likewise generated by inserting the cognate DNA amplicons into the same restriction sites ( Figure 1A). After cloning in E. coli NEB5a, these plasmids were integrated in the genome of L. monocytogenes strains LL195 at the tRNA Arg locus as previously described (Lauer et al., 2002).
For allelic replacement at the hlyA locus ( Figure S3A), pMAD-∆hlyA::FAST was created by amplifying three partially overlapping fragments by PCR: One thousand base pairs (bp) upstream (plcA gene) and downstream (mpl gene) of the hlyA open reading frame in the EGD-e genome were amplified, respectively, with oAL981-2 and oAL976-7, while the FAST-Myc open reading frame was amplified from pAD-FAST with oAL983-75. These fragments were inserted into the pMAD vector (Trieu-Cuot et al., 1991), between the SalI and BglII restriction sites by Gibson Assembly, using the NEBuilder HiFi DNA Assembly Cloning Kit (New England BioLabs). pMAD-hlyA-FAST containing the last 1000 bp of hlyA fused with the FAST sequence, a Myc tag and one thousand bp downstream of hlyA was likewise generated by inserting the cognate DNA amplicons into the same restriction sites in pMAD. Allelic replacements of the hlyA open reading frame by these constructs in the genomes of L. monocytogenes strains LL195 and EGD-e were obtained as previously described (Trieu-Cuot et al., 1991). For complementation purposes in haemolysis assays, a simple in-frame deletion mutant of the hlyA gene was also created using the pMAD backbone.

The complete lists of bacterial strains and oligonucleotides used in this work are supplied as Supplementary
Tables S1 and S2, respectively.

Bacterial total extracts or secreted protein analysis
Bacterial total extracts or culture supernatants were recovered from 1 ml of Lm strains grown to an OD600nm of 2.0 in BHI medium at 37°C as previously described (Lebreton et al., 2011).
Total bacterial extracts of Sf were prepared by boiling for 2 ´ 10 min at 95°C in 100 μl of Laemmli sample buffer (SB 1X) the bacterial pellets obtained by centrifugation of 1 ml of each strain grown to an OD600nm of 2.0 in TCS medium at 37°C. For assessment of secretion leakage prior to T3SS induction, 2 ml of Sf culture supernatants were collected, precipitated with 16% trichloroacetic acid (TCA), and centrifuged for 30 min at 16,000 ´ g at 4°C. Protein pellets were washed twice in acetone before resuspension in 50 μl of SB 1X. For induction of secretion, Sf were resuspended in 0.6 ml phosphate buffered saline (PBS) containing 1 mg/ml of Congo red at a final OD600nm of 40, and incubated at 37°C for 45 min. Bacteria were eliminated by centrifugation; 100 μl of supernatant were collected and mixed with 33 μl of SB 4X for SDS-PAGE separation.
The remainder supernatant was TCA-precipitated and resuspended in 50 μl SB 1X as above.
10 μl of each sample were separated on 4-15% Mini-Protean TGX gels (Bio-Rad) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For immunoblots, after transfer on nitrocellulose membrane (Amersham) using PierceG2 Fast Blotter, proteins were probed with anti-Myc mouse monoclonal antibody #9E10 (sc-40, Santa Cruz Biotechnology) at a 1:400 dilution in PBS supplemented with 0.05% tween-20 and 5% skimmed milk powder, followed by secondary hybridization with anti-Mouse IgG-heavy and light chain Antibody (Bethyl) at a 1:50 000 dilution in the same buffer. Signals were detected using Pierce ECL Plus Western Blotting Substrate and a Las4000 imager (GE Healthcare). Staining with colloidal Coomassie Brillant blue G-250 was performed as previously described (Neuhoff et al., 1988).

Haemolysis assay
The supernatants of overnight-grown cultures of Lm in BHI medium were recovered by centrifugation for 1 min at 6,000 ´ g followed by filtration through 0.2-μm pore filters, in order to eliminate bacteria. Serial twofold dilutions of these supernatants were performed in round-bottom, clear, 96-well plates (100 μl final volume per well) using as a diluent PBS, the pH of which was adjusted to 5.6, and supplemented with 0.1% bovine serum albumin (BSA). Erythrocytes from defibrinated mice blood were washed twice in PBS pH 6.4 and diluted 1/10 th in PBS pH 5.6. 50 μl of this suspension was added to each one of the wells containing diluted culture supernatants. After 30 min of incubation at 37°C, the plates were centrifuged for 10 min at 430 ´ g and haemolytic titres were calculated as the reciprocal of the dilution for which 50% of haemolysis was observed (Roche et al., 2001).

Fluorescence measurement on culture supernatants
Lm were grown overnight in BHI, washed and diluted to 1:10 th in iLSM, and then grown for 6 h at 37°C, The standard curve for FAST fluorescence quantification was performed by diluting, in control medium, known amounts of recombinant FAST produced in E. coli Rosetta(DE3)pLysS as previously described (Plamont et al., 2016).
Each experiment was reproduced three times independently.

Infection and transfection of epithelial cells
Infections of intestinal epithelial cells were performed in the LoVo cell line originating from colon adenocarcinoma (ATCC Cat# CCL-229, RRID: CVCL_0399), grown in Ham's F-12K medium supplemented with 10% FBS, following ATCC recommendations. The Caco-2 epithelial cell line (ATCC Cat# HTB-37, RRID: CVCL_0025), also from colon adenocarcinoma, was used as a control that LRVs were not a specificity of LoVo cells. All cells were cultured at 37°C in a humidified atmosphere containing 5% CO2. For live microscopy, cells were seeded on Ibidi μslides 72 h prior to infection at a density of 10 5 cells/ml, in 300 μg/ml of culture medium. When needed, cells were transfected 24 h before infection with pEYFP-C1-CBD expressing YFP-CBD (Henry et al., 2006), using Lipofectamine LTX (Invitrogen) and 1 μg/ml of plasmid, according to the manufacturer's specifications.
Lm strains were grown in BHI medium until they reached early stationary phase (OD600 of 2 to 3), washed in pre-warmed D-MEM, and then diluted in culture medium without serum to achieve a multiplicity of infection (MOI) of 5 (for long-term infections) to 30 (for short-term infections). Except for short-term imaging when bacterial entry was monitored under the microscope, after 30 min of bacteria-cell contact the inoculum was washed away by washing cells twice with serum-free medium containing 40 μg/ml of gentamicin, then the medium was replaced by complete culture medium without phenol red containing 25 μg/ml in order to kill extracellular bacteria. For live cell imaging, preparations were observed with a Nikon Ti PFS microscope coupled to a spinning disk confocal device (CSU-XI-A1, Yokogawa), connected to a cooled EM-CCD camera (Evolve, Photometrics), and equipped with a Cube for temperature control and a Brick gas mixed for a CO2 and humidity control (Life Imaging Services). Image acquisition and microscope control were actuated with the MetaMorph software (Molecular Devices, RRID:SCR_002368). Fluorescent illumination was driven by three lasers, of wavelength 491 nm for eGFP, YFP of FAST, 561 nm for mCherry, and 635 nm for SiR-actin. Images were acquired with apochromat 63x objective lenses (NA 1.4) in 1 μm step-Z-stacks. Acquisition parameters were similar for all samples of an experiment. For snapshot display, maximum intensity signals from 16 successive stacks (i.e. 16 μm slices) were integrated with Fiji (RRID:SCR_002285). Each picture or video is representative of the population observed in three independent experiments.

Live fluorescence microscopy of infected cells
For measurement of FAST accumulation in cells, images were first z-projected by maximum intensity, then fluorescence intensities were quantified on regions of fixed areas using Fiji. Vacuole tracking and bacterial growth rate calculations were performed using Matlab scripts (RRID:SCR_001622) tailored to each purpose as detailed below.

Tracking of primary vacuoles in short term infection assays
The slices of the z-stack obtained from spinning confocal imaging were projected onto a single plane (maximal projection). Fluorescent vacuoles were tracked using the plugin TrackMate in Fiji. The time at which tracks began during the infection was used to compute the time of Lm entry into LoVo cells. We then reconstructed the cumulative distribution of entries as a function of time and the entry rate was obtained by fitting the slope of the linear cumulative distribution of entries with time. Finally, the distribution of residence times in primary vacuoles was computed from the statistics of track length.

Tracking of LRVs in long-term infection assays
At 2 h p.i., Ibidi μslides were mounted on a confocal spinning disc microscope for observations. The mCherry signal labelling the bacterial cytoplasm was used to segment the volume of bacteria. Given the good signal-to-noise ratio of mCherry images, we performed a direct Otsu-thresholding algorithm on the mCherry stacks to obtain the 3D segmentation of bacteria. We then used MatLab routines to track objects based on their size and their location. To measure LLO-FAST signals in primary vacuoles, we applied on the FAST images the binary masks retrieved from mCherry segmentation and computed the average FAST signal in each mask.
The fraction of the primary vacuoles into which Lm replicated was computed as the ratio of the number of tracked vacuoles that at least doubled their size during the course of the movie (12 h) to the initial number of bacteria. The growth rates of bacteria inside LRVs were computed by fitting the dynamics of segmented mCherry volumes to an exponential function.

Immunofluorescence or LysoTracker staining of infected cells
LoVo cells were seeded 48 h before infection in 24-well plates containing 12 mm diameter coverslips precoated with poly-L-lysine. Infection with bacteria expressing mCherry (for immunofluorescence experiments) or eGFP (for LysoTracker staining) was performed as described above, using a MOI of 30, except that plates were centrifuged for 1 min at 200 ´ g after addition of the inoculum in order to synchronise bacteria-cell contacts. 3 h p.i., cells were washed in pre-warmed PBS, fixed 20 min with 4% paraformaldehyde in PBS, then permeabilized for 5 min at room temperature with 0.5% Triton X-100 in PBS, and blocked for 5 min in PBS buffer containing 2% bovine serum albumin (BSA, Sigma). Incubation with primary antibodies in PBS buffer, 1 % BSA was performed for 1 h, followed by three PBS washes, and incubation with the Alexa Fluor 647-conjugated secondary anti-rabbit antibody (Molecular probes Cat# A21245, RRID:AB_141775, 2 μg/μl),

Acknowledgements
We are grateful to Marie-Aude Plamont, Vinko Besic, Sebastian Rupp and Alison Tebo for their precious experimental assistance and eagerness to help solve technical issues. We thank Lionel Schiavolin and Didier Filopon for providing source strains and practical advice regarding Sf experiments. We thank the IBENS imaging facility for maintaining access to microscopy equipment that was instrumental to this work, and providing expert support whenever needed. We are indebted to the IBENS animal facility for kindly supplying the mice blood used in haemolysis assays. We thank Jost Enninga for unfailing proof of enthusiasm and insightful discussion.  Shen, A., Higgins, D.E., 2005. The 5' untranslated region-mediated enhancement of intracellular listeriolysin O production is required for Listeria monocytogenes pathogenicity. Mol Microbiol 57, 1460-1473. doi:10.1111/j.1365-2958     Observations were performed on an inverted spinning disk microscope. Scale bars, 5 μm.

Supplementary materials
Supplementary materials include Movies S1 to S5, Figures S1 to S5 and Tables S1 to S2.

Movies
Movie S1. Accumulation of secreted FAST in the cytoplasm of infected cells.

Movie S4. Observation of the decoration of LRVs by LLO-FAST in Listeria cells infected by
Lm EGD-e.
LoVo cells infected with Lm EGD-e expressing both mCherry and LLO-FAST were observed between 2 and 9 h post-infection by spinning disk microscopy. Orange, FAST channel; cyan, mCherry channel; purple, SiRactin channel. Scale bar, 5 μm.
Movie S5. Imaging of the differential labelling by CBD-YFP of the cytosolic versus intravacuolar populations of intracellular bacteria.
LoVo cells were transfected with pEYFP-CBD 24 h being infected with Lm expressing mCherry, then imaged by spinning disk microscopy from 2 to 8 h p.i. Yellow, CBD-YFP channel; cyan, mCherry channel. Scale bar,   (B) Haemolytic properties of the Listeria monocytogenes strains producing FAST-or eGFP-tagged fusion LLO used in this study. The haemolytic titre measured for the strain where LLO was C-terminally tagged with FAST-Myc at the hlyA locus (hlyA-FAST) did not differ from that of the WT Lm strain. Haemolytic titres were enhanced for ∆hlyA deletion strains that had been complemented by hlyA fusion genes under control of the strong, constitutive PHYPER promoter in the pAD vector. Fusion with FAST-Myc or eGFP-Myc did not affect haemolytic properties, compared to a simple fusion with Myc. None of these strains reached the intense haemolytic properties of the prfA* strain, where the expression of Lm virulence genes (including hlyA) is deregulated, due to the constitutive activity of the transcriptional activator PrfA (Ripio et al., 1997). The haemolytic titre of the ∆hlyA strain being null, it was not represented on this graph. ANOVA was used for statistical testing. ns, non-significant; ***, p < 0.001.
(C) Time-course of replication of mCherry-expressing Lm inside a LRV for the WT, the prfA* and the ∆hlyA strains during the infection of LoVo cells. Scale bar, 5 μm.