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

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.


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 40 invasive bacteria with multiple abilities, such as that of crossing cellular 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 Chlamydia trachomatis, Brucella abortus or Legionella pneumophila perturb the maturation and rearrange the properties of vacuoles, thereby 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 [12].
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 70 infection [9]. Upon encapsulation into the internalisation vacuoles together with invading bacteria, the fluorescent dye stained the intravacuolar space until it broke down. However, this method required a washing step after bacterial entry in order to remove the unwanted background due to the high extracellular amount of the dye. This washing step thus prevented the observation of the first 10 minutes of the infection dynamics. Alternative strategies were based on the assessment of vacuole rupture events and bacterial access to the host 75 cytosol using fluorescent sensors. For instance, galectin-3 was shown to label membrane remnants of damaged vacuoles and thereby allowed the spotting of vacuole lysis [13]. 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 [5,14]. Cytoplasmic FRET probes that are cleaved by a b-lactamase secreted by invasive bacteria have also been described as efficient reporters of average vacuole rupture time at a cellular 80 scale [15,16]. Although 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), they did not allow a precise recording of the onset of entry events, which prevented their use for establishing the distribution of Lm residence time in entry vacuoles with accuracy. These constraints have limited the possibilities of refined quantitative comparisons of variations in intravacuolar residence times between different conditions. 85 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 integrity of the vacuole membrane was disrupted. We devised a strategy based on tagging proteins secreted by bacteria with the FAST reporter system [17]. FAST is a 14-90 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 rapid folding kinetics, the reversible binding of fluorogens together with good brightness and photostability made this system an ideal candidate for tagging secreted proteins such as LLO and imaging them in real time. 95 Using live imaging of FAST-tagged proteins, 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, the prolonged residence of Lm inside vacuoles was observed in cells infected with wild type (WT) Lm as well as with a hlyA deletion strain. The secretion of LLO allowed Lm to proliferate actively inside these compartments, 100 suggesting that besides its role in vacuole escape, LLO may contribute to setting up an intravacuolar niche permissive for Lm replication in epithelial cells.

Fluorescent tagging with FAST of proteins secreted by Listeria monocytogenes
With the aim of detecting proteins that were secreted by intracellular bacteria into their host cells in live- 105 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 ( Fig 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-110 eGFP constructs), or to full-length LLO (LLO-FAST, LLO-eGFP and untagged LLO constructs), a classical Sec substrate. A Myc tag in the 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 16h cultures in brain heart infusion (BHI) (S1 Fig). All transgenes were efficiently expressed by Lm, even though 115 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 (S1C, D Fig). 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 through the 120 general secretion pathway.
To assess whether the FAST reporter system remained fluorescent after secretion, we quantified the fluorescence signals in the filtered culture medium of bacteria grown for 6 h in Listeria synthetic medium (LSM) (Fig 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. By calibrating fluorescence measurements with a standard curve of 125 known FAST concentrations diluted in the same medium, we estimated the concentration of secreted tagged proteins; that of SP-FAST reached 325 ± 55 nM, and that of LLO-FAST was 28 ± 6 nM. In contrast, fluorescence levels in the culture medium of strains producing non-secreted FAST remained low, indicating that the release of fluorescent proteins in the culture medium due to bacterial lysis was minor. We conclude that FAST-labelled proteins retained their fluorescent properties after undergoing secretion through Sec.

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Diverse attempts by others in Gram-negative bacteria [18] 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-also 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 (S2 Fig). A consistent proportion of eGFP undergoing Secdependent secretion was thus able to acquire its mature fold in bacterial culture medium, at least in conditions where it was not fused to a bacterial effector and in LSM.

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 the Cterminal ends of the effectors OspF and IpaB with FAST-Myc (Fig 1C), which are translocated upon adhesion of Sf to host cells [19]. Bacterial total extracts and culture supernatant fractions were recovered from 16-h cultures in M9 medium, with or without stimulation of type-III dependent secretion by addition of Congo red.

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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 (S3A Fig). The secretion of both tagged effectors was constitutive when using a ∆ipaD mutant strain for which translocation lacks gating controls [20] (S3B Fig). 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 16-h bacterial cultures in 150 M9 medium (Fig 1D). Fluorescence levels were consistently higher with this constitutively secreting strain ∆ipaD 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 pass through the needle of the TS33, and keep fluorescent properties after secretion at least when gating controls are lacking.

FAST-tagging of secreted Listeria effectors for live fluorescence microscopy
We next investigated whether the FAST reporter system was suited for detecting proteins secreted into the cytoplasm of host cells by real-time microscopy 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 (Fig 2, S2 Movie). FAST fluorescence labelled uniformly the cytoplasm of infected cells and increased 160 over time (Fig 2A). At 562 nm (the emission wavelength specific for FAST:HBR-3,5DM), fluorescent signals accumulated in cells infected with a strain producing SP-FAST, and not with a control isogenic strain that constitutively expressed mCherry (Fig 2B). In infected cells, measured fluorescence intensities-which reflects the intracellular concentration of SP-FAST-increased exponentially with time ( Fig 2C), likely mirroring the exponential growth of Lm in the host cytosol. After several hours of signal accumulation, the 165 intracellular fluorescence dropped abruptly. This corresponded to a sudden shrinkage of infected cells, probably resulting from their death and from the concomitant permeation of their membranes. For each cell, we fitted the dynamics of fluorescent signals to an exponential curve as shown in Fig 2C (black curve), measured the rates of fluorescence increase for each exponential curve, calculated fluorescence doubling times (t = !"# $ ), and then plotted their distribution ( Fig 2D). The mean doubling time of FAST signals was 170 106.7 ± 41.9 min (n = 39). This value, which represents the characteristic time for SP-FAST accumulation in infected cells, was comparable to the mean doubling time of bacteria (94.7 ± 7.9 min, n = 4) measured for mCherry-labelled bacteria in similar conditions of infection and illumination (S4 Fig). Altogether, the secretion of FAST into host cells allowed a quantitative monitoring of infection progression by live imaging of individual cells.

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 clearly 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 180 secreted FAST molecules in a reduced volume would allow their detection and tracking until the disruption of vacuole membranes, thereby providing an accurate measurement of individual vacuole lifetimes ( Fig 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, most likely when vacuoles ruptured (Fig 3B, Movie 2). 185 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 (Fig 3C). 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, S5A Fig) and also expressed mCherry constitutively. The growth rates of these two strains in BHI were undistinguishable (S5B Fig). The median value for the residence time of the 190 WT strain was 12.7 ± 0.7 min (Fig 3C). When using the ∆hlyA::SP-FAST strain, the distribution of residence times was significantly shifted compared to the WT (p = 0.0191). The median residence time was 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 [11]. Unexpectedly, a consistent proportion of the entry vacuoles lasted for more than one hour (12.0 % for 195 the WT strain; 14.8 % for the ∆hlyA mutant), and intact vacuoles were still observed 3 h p.i. (4.6 % for the WT strain; 6.2 % for the ∆hlyA mutant) (Fig 3C). The fact that the WT strain remained entrapped in vacuoles 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 200 LLO fused to a FAST tag was detected in vacuoles from 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 vacuoles, we engineered a Lm strain where LLO was C-terminally fused with FAST at the endogenous hlyA locus (S5A Fig).
In this strain, the fluorescence of FAST reported not only for LLO secretion and localisation, but also for hlyA 205 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 (S5C Fig); the production, secretion and activity as a cytolysin of LLO are thus quantitatively and qualitatively preserved after C-terminal fusion with FAST.

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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 vacuoles for several hours before the enclosed structure of LLO-labelled membranes eventually disrupted and bacteria dispersed into the cytosol (  3) and the European Lm reference strain EGD-e (lineage II, ST9) (S6A Fig, Movie 4), indicating that this property was not specific to the hypervirulent clone LL195. Likewise, the proliferation of Lm inside long-term vacuoles decorated with LLO-FAST was observed in Caco-2 cells, suggesting that LoVo cells were not the only epithelial niche allowing Lm to replicate inside endomembrane compartments (S6B Fig). Because these vacuoles were reminiscent of the SLAPs previously described in macrophages [12], we will refer to them as 225 eSLAPs (for epithelial SLAP-like vacuoles) hereafter.
By tracking vacuoles and segmenting the mCherry fluorescence signals, we computed the volume occupied by intravacuolar bacteria, which is proportional to their number, and thereby determined the growth rate of WT Lm in eSLAPs (S7A Fig). Segmenting mCherry-labelled bacteria also allowed the measurement of growth rates of free bacteria in the cytosol (S8 Fig). Like cytosolic growth, intravacuolar growth was exponential, 230 with a mean doubling time (102.2 ± 36.3, n = 18) similar to that of cytosolic bacteria (96.3 ± 13.0, n = 7) ( Fig 4B). This doubling time was consistently faster than that previously described in SLAPS, which was in the range of 8 h [12].

Role of listeriolysin O in the long-term intravacuolar residence and replication of Listeria
Our above-mentioned results showed that LLO-FAST was secreted by Lm and functional, and also that it was present in eSLAPs, which is likely to permeate their membranes. However, despite the presence of LLO, the integrity of eSLAPs was preserved over several hours, and intravacuolar replication of Lm occurred without vacuole rupture. To determine whether LLO concentration influenced Lm residence time in eSLAPs, we took advantage of the LLO-FAST reporter strain in order to measure the variability in LLO abundance in vacuoles. LLO-FAST signals measured in eSLAPs displayed a broad spectrum of dynamics, indicating that eSLAP formation and duration were independent of the amounts of secreted LLO (S7B Fig). In some vacuoles, LLO-FAST accumulated linearly over time, while others displayed large-scale fluctuations in signals that may reflect variations in LLO synthesis, secretion, degradation, leakage from vacuole or membrane repair. Some eSLAPs yielded a strong signal while others displayed low levels of decoration by LLO. The lifetime of eSLAPs was correlated with neither the average nor the maximal level of LLO-FAST concentration in eSLAPs (S7C, D Fig), suggesting that LLO concentration had a limited influence on the probability of Lm escape from these structures.
To further assess the effects of LLO concentration on the stability of eSLAPS, we tracked intravacuolar bacteria for WT, ∆hlyA and prfA* mutant strains (Fig 4C, S9 Fig). When using the hlyA deletion strain, lasting vacuoles were observed (Fig 3C, 4C, S9 Fig). However, ∆hlyA bacteria were unable to proliferate inside these 250 long-lived vacuoles (Fig 4C, S9 Fig). Similar to SLAPs, eSLAPs thus required that bacteria secreted LLO to allow intravacuolar growth.
To examine whether the activity of LLO as a pore-forming toxin was required for bacterial growth in eSLAPs, we carried out the same experiment using a strain with a W492A point mutation in LLO that had been previously described to almost abolish haemolytic activity [21]. The introduction of this mutation in 255 chromosomal hlyA-FAST in the LL195 background resulted in a strain that retained ~1 to 3 % of the haemolytic titres of the isogenic WT strain (S5C Fig). These bacteria were still able to grow in vacuoles (S9 Fig), suggesting that either the pore-forming toxin activity of LLO was itself dispensable for allowing intravacuolar growth, or the residual activity of the toxin was enough to support growth.
Conversely, to assess the effects of increased LLO production, we used a prfA* mutant strain to investigate 260 the outcome of LLO overexpression. The prfA* allele encodes a PrfA variant with a G145S substitution that has been previously described to be constitutively active, and to lead to the strong overexpression of PrfAdependent virulence genes, including that of hlyA [22]. Accordingly, the in-vitro haemolytic titre of the LL195 prfA* strain was fifty-fold higher than that of the isogenic WT strain, indicative of LLO hyperproduction (S5C Fig). Using WT or prfA* reporter strains where eGFP was inserted by allelic replacement under control of the 265 endogenous PhlyA promoter, we measured that eGFP expression was on average 6.7-fold higher in the prfA* strain than in the WT strain at 1 h p.i., and 2.7-fold higher at 3 h p.i. (S10 Fig). In our experimental model, the prfA* mutation thus also led to overexpression of hlyA by intracellular bacteria, at least in the first hours of infection when eSLAPs start being formed by intravacuolar bacteria. Despite higher levels of hlyA expression, eSLAPs were still detectable for the prfA* strain, indicating that increased secretion of the pore-forming toxin 270 did not hamper the ability of Lm to reside and multiply inside vacuoles during several hours. This feature contrasts with SLAPs, which formed in phagocytes only when the expression of hlyA was moderate [12]. LLO quantity did not influence intravacuolar bacterial growth, since the prfA* strain replicated at a similar rate as the WT strain in eSLAPs ( Fig 4C). Consistently, the growth rate of LLO-FAST-secreting bacteria in eSLAPs was correlated with neither the average nor the maximal level of LLO-FAST fluorescence intensity (S7E, F 275 Fig). Nevertheless, we observed by live-cell imaging that the escape of the prfA* strain from eSLAPs occurred earlier than for the WT strain ( Fig 4C, S9 Fig). In agreement with this observation, the ratio of eSLAPs to the initial number of entry events was lower when cells were infected with the prfA* strain than with the WT strain ( Fig 4D). This higher probability of vacuole escape for the prfA* strain suggests that very a high concentration of LLO exerts a mild destabilising effect on the integrity and duration of eSLAPs, though it does 280 not preclude their formation.
Altogether, our results suggest that the secretion of LLO is required for proliferation of Lm in eSLAPs, but that its concentration and overall activity exert only a minor influence upon eSLAP lifetime and on the ability of bacteria to grow inside. 285 The eSLAPs in which Lm replicated (Fig 4, Movies 3, 4) likely originated from internalisation vacuoles from which bacteria had failed to escape (Fig 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 host cytoplasm. To assess whether eSLAPs resulted from primary vacuoles, we monitored the intravacuolar stages of mCherry-expressing bacteria in LoVo cells transfected with the YFP-CBD fusion 290 protein reporter [14]. This reporter has been previously described to specifically label the surface of bacteria once 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 eSLAPs remained unlabelled with YFP-CBD until the vacuole ruptured and bacteria dispersed throughout the cell ( Fig  out the possibility that bacteria became entrapped into secondary vacuoles by canonical autophagy or cell-tocell spread after a first exposure to the host cell cytosol, and thereby confirmed that eSLAPs where Lm replicated originated from internalisation vacuoles.

Origin and properties of Listeria long residence vacuoles in epithelial cells
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 300 epithelial cells rather than in phagocytes. We thus carried out a molecular characterization of this intravacuolar replication niche in order to analyse whether it had typical features of endosomal, lysosomal and/or noncanonical autophagy-derived compartments. By immunofluorescence staining of LoVo cells infected with mCherry-expressing Lm for 3 hours, we observed that the vacuoles containing several bacteria were negative for the early endosomal marker Rab5 (9% of colocalisation, n = 22), while they were positive for the late Altogether, we conclude that eSLAPs display molecular characteristics highly reminiscent of SLAPs, although 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 315 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 an intravacuolar replication 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 [14]. LLO localisation at vacuole membranes, or more recently in bacterial-derived membrane vesicles, was first observed by electron microscopy using immunogold labelling [23,24]. However, the precise dynamics of 325 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 [25]. Nevertheless, ectopic expression by host cells cannot mimic the concentrations, location, and insertion into membranes from 330 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 data supporting that FAST-tagged effectors can also be efficiently secreted through the T3SS of S. flexneri.
In recent years, several strategies have emerged for fluorescent labelling of Sec-or T3SS-dependent substrates [26]. 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 [27,28]; 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 345 systems. Superfolder GFP (sfGFP) or its derivative rsFolder have been successfully used for labelling E. coli periplasmic proteins exported through the Sec pathway [18,29], but to our knowledge has not been applied yet for other bacterial systems or in the context of host-pathogen interactions. Other fluorescent tags such as FlAsH and phiLOV were successfully used for monitoring the secretion of Sf T3SS-dependent effectors [30,31]. Nevertheless, the toxicity in eukaryotic cells of the biarsenite dye used for FlAsH labelling and the rather 350 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 [32,33] as well as (d) its rapid and reversible folding dynamics allowing transport through the T3SS (compared with GFP-derived probes); (e) its reasonable 355 brightness and fast maturation time (compared with PhiLOV). In addition, FAST-labelled proteins can be imaged at different wavelengths between 540 and 600 nm by selecting the appropriate fluorogen [34], thereby providing users with flexibility in the choice of other fluorescent reporters in co-localisation studies. Redshifted fluorogens also limit the toxicity of certain wavelength for bacteria when performing long-term imaging, and membrane-impermeant fluorogens offer the possibility to discriminate between intracellular and 360 extracellular proteins [35], for instance when addressing the localisation of bacterial effectors that anchor to the bacterial cell wall or to membranes [36].
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. 365 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 epithelial SLAP-like vacuoles (eSLAPs), which are positively labelled by LLO-FAST (Fig 6). After several hours of intravacuolar residence and growth, eSLAPs eventually break open and bacteria resume a canonical cytosolic lifestyle.

eSLAPs, an alternative replication niche for Listeria monocytogenes in epithelial cells
The decoration of eSLAPs by LC3, Rab7 and LAMP1 as well as their neutral pH are reminiscent of the 370 SLAPs (Spacious Listeria-containing Phagosomes) previously described in phagocytes [12], and which derive from LAP (LC3-associated phagocytosis) [37]. Upon infection by Lm, we propose a model for the formation of replicative eSLAPs, analogous to the current model of SLAP formation (Fig 6). The entrapment of Lm inside internalisation vacuoles could result in two distinct fates. (A) In the classically-described pathway, the coordinated actions of LLO, PlcA and PlcB result in a rapid disruption of the vacuole and escape of bacteria 375 into the cytoplasm, where they can start replicating and polymerising actin. (B) In the second scenario, a proportion of internalisation vacuoles would undergo LC3 lipidation in addition to their maturation attested by decoration with Rab7, as well as fusion with lysosomes as suggested by LAMP1 labelling.
Whereas the eSLAPs observed in LoVo cells display similarities with SLAPs, they are notably distinct from LisCVs, which are an intravacuolar persistence niche of Lm recently described in human hepatocytes and 380 trophoblast cells [38]. Contrary to SLAPs and eSLAPs, LisCVs do not derive from primary vacuoles. Instead, they form late in the intracellular cycle of Lm by recapture of bacteria that have lost ActA-dependent motility. Indeed, bacteria found in LisCVs are labelled with YFP-CBD, while the bacteria we observe in eSLAPs are not. Moreover, whereas eSLAPs are lipidated by LC3, LisCVs are not. Last, Lm replicates in eSLAPs, whereas it adopts a viable but non-culturable state in LisCVs and does not grow. Altogether, though occurring in epithelial cells, the features we describe for eSLAPs are in agreement with compartments similar to SLAPs, and distinct from LisCVs. Our observations that even low LLO activity allows Lm residence and replication in vacuoles is consistent with the previous report by Birmingham et al. showing that reduced hlyA expression allowed slow replication in macrophage SLAPs [12]. However, the replication of Lm inside eSLAPs is significantly faster than the 8 hours of doubling time reported in SLAPs [12], perhaps due to a lower 390 bactericidal capacity of the epithelial niche compared with phagocytes. Membrane permeation by LLO might also attenuate the bactericidal properties of eSLAPs, and/or allow nutrient uptake through the permeated membrane, thereby promoting bacterial replication.

Conclusion
Together with LisCVs and SLAPs, eSLAPs enrich the palette of Lm intravacuolar lifestyles that can 395 establish in various cells types. Apprehending the importance of eSLAPs in the context of in vivo infections prompts future investigation. Indeed, whilst intravacuolar lifestyles impose constraints on motility or nutrient uptake, these compartments might provide shelter from cytosolic surveillance mechanisms such as autophagy and RIG-I-dependent activation of type-I interferon signalling, or favour chronic forms of infections by dampening cell-to-cell spread within tissues. Conversely, delayed residence within vacuoles could promote 400 recognition of Listeria pathogen-associated molecular patterns by endosomal Toll-like receptors and activate NF-kB-dependent inflammatory pathways. Prolonged exposure to the intravacuolar environment could also tune the expression of Lm virulence genes. Deciphering the extent to which these intravacuolar niches influence the balance between bacterial fitness and host defences becomes critical to better appreciate longterm 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 of Listeria monocytogenes LL195 (lineage I, ST1) [39] for most of the experiments involving Lm, and Shigella 410 flexneri M90T [40] for experiments on Sf T3SS-dependent secretion. Lm reference strain EGD-e (lineage 2, ST9) [41] (lineage II, ST9) was also used as a control that the observed eSLAPs were not specific to LL195. All strains were grown at 37°C under shaking at 190 rpm in Luria Bertani (LB) medium for E. coli, in LB or tryptic soy broth (TSB) for Sf, in brain hear infusion (BHI) or Listeria synthetic medium (LSM) [42] for Lm. Whenever required, media were supplemented with antibiotics for plasmid selection (chloramphenicol, 415 35 μg/ml for E. coli; 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 (S12A For plasmid constructions in the pAD vector derived from the pPL2 backbone [43,44], the 5'-UTRhlyA-SPhlyA-FAST-Myc fusion was amplified with primers oAL543-4, the sequence of the Lm hlyA gene encoding 425 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) [44] 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 (Fig 1A). pAD-FAST, pAD-eGFP, pAD-SP-eGFP, pAD-hlyA, pAD-hlyA-FAST and pAD-hlyA-eGFP, all containing the 5'-430 UTR of hlyA and a Myc tag, were likewise generated by inserting the cognate DNA amplicons into the same restriction sites (Fig 1A). After cloning in E. coli NEB5a, these plasmids were integrated in the genome of L. monocytogenes strains LL195 at the tRNA Arg locus by electroporation as previously described [43]. The pHpPL3-mCherry plasmid was introduced at the tRNA Arg locus by conjugation [45]. in the genomes of L. monocytogenes strains LL195 and EGD-e were obtained as previously described [46]. 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 Tables S1 and 455 S2, respectively.

Analysis of protein contents in bacterial total extracts or culture media
Bacterial total extracts or culture supernatants were recovered from 1 ml of Lm strains grown to an OD600nm of 2.0 in BHI at 37°C as previously described [48]. Coomassie Brilliant blue G-250 or by immunoblotting. 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 475 ECL Plus Western Blotting Substrate and a Las4000 imager (GE Healthcare).

Fluorescence measurements on culture supernatants
Lm were grown for 16 h in BHI, washed and diluted to 1:10 in Listeria synthetic medium (LSM), and then grown for 6 h at 37°C, 180 rpm. Likewise, for secretion by Sf a culture in TSB was diluted to 1:10 th in M9 medium supplemented with 0.2% glucose and 10 μg/ml nicotic acid. From 1 ml of culture, bacterial pellets for eGFP. Background fluorescence was measured on culture media from negative control strains (Lm LL195 [pAD-LLO] or Sf M90T ∆ipaD) and subtracted to each sample. The standard curve linking FAST fluorescence to its concentration was performed by diluting, in control medium corresponding to a culture of the corresponding negative control strain, known amounts of recombinant FAST produced in E. coli 490 Rosetta(DE3)pLysS as previously described [17]. This enabled the calculation of fluorescent FAST-tagged proteins released in each culture.
As a negative control that the fluorescence was due to the formation of the FAST-HBR-3,5DM complexes, the fluorescence was also measured on samples mixed with 20 μl of PBS instead of PBS containing HBR-3,5DM. No fluorescence was detected above that of the control culture media.

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For normalisation between measurements for FAST-and eGFP-tagged proteins, fluorescence intensities measured in filtered culture media were expressed relatively to the fluorescence measured for a suspension of OD600nm = 1 of Lm constitutively expressing either non-secreted FAST, or eGFP. The fluorescence intensities emitted by each one of these reference suspensions were fixed arbitrarily to 100 A.U.
Each experiment was reproduced three times independently. Statistical significance was assessed by two-500 tailed Student's t-tests with equal variance assumption, on the results of three independent experiments.

Haemolysis assay
The supernatants of 16-h cultures of Lm in BHI 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 two-fold dilutions of these supernatants were performed in round-bottom, clear, 96-well plates (100 μl final volume per well) using 505 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 [49]. Two-way 510 ANOVA on log2-transformed haemolytic titres followed by post-hoc Tukey test was used for statistical testing between conditions.

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). The Caco-2 epithelial cell line (ATCC Cat# 515 HTB-37, RRID: CVCL_0025), also from colon adenocarcinoma, was used as a control that eSLAPs were not a specificity of LoVo cells. All cells were cultured in Dulbecco′s Modified Eagle's medium (D-MEM) supplemented with 10% FBS, 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 ml of culture medium. When needed, cells were transfected 24 h before infection with pEYFP-C1-CBD expressing YFP-520 CBD [14], using Lipofectamine LTX (Invitrogen) and 1 μg/ml of plasmid, according to the manufacturer's specifications.
Lm strains were grown in BHI until they reached early stationary phase (OD600 of 2 to 3), washed in prewarmed 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 525 bacterial entry was monitored under the microscope, after 1 h of bacteria-cell contact the inoculum was washed away twice with serum-free medium containing 40 μg/ml gentamicin, then the medium was replaced by complete culture medium without phenol red containing 25 μg/ml gentamicin in order to kill extracellular bacteria. For live cell imaging, preparations were observed with a Nikon Ti PFS microscope coupled to a spinning 535 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 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 or FAST, 561 nm for mCherry, and 635 nm for SiR-actin. Images were 540 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.

In-situ growth measurements of mCherry-labelled bacteria
We performed for each time point an Otsu-thresholding on the entire z-stack of mCherry images in order 555 to obtain the 3D segmentation of bacteria. Although the segmentation was not able to isolate individual bacteria in dense regions, it yielded the total volume occupied by bacteria in the field of view for each frame. The growth rate of mCherry bacteria was measured by fitting the total volume of segmented objects ( ) to an exponential curve ( ) = % $& , with the growth rate and % the initial volume. The doubling time t was then computed according to the following formula: t = !"# $ . Image computing was performed using MatLab 560 RRID:SCR_001622.

Tracking of primary vacuoles in short term infection assays
The slices of the z-stacks 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. The distribution 565 of residence times in primary vacuoles was computed from the statistics of track lengths. Statistical significance was assessed by two-tailed Student's t-test on the two distributions, with equal variance assumption. The interpolated median ( ) lifetime of SP-FAST-labelled vacuoles was calculated from the raw distributions as follows: , where is the median, is the time interval, and ( , ! and . are the number of events greater, lower or equal to , respectively. The accuracy of the interpolated median 570 estimate was assessed by boostrapping analysis (100 random samplings for each distribution, yielding 100 estimates of for each condition) followed by two-tailed Student's t-test on the distributions of the interpolated medians (p << 10 -5 ).

Tracking of eSLAPs in long-term infection assays
At 2 h p.i., Ibidi μslides were mounted on a confocal spinning disc microscope for time-lapse observations.

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Pixel size was 250 x 250 nm and step size in z was 1 µm. For each time point taken every 5 or 15 minutes, we recorded a z-stack of fluorescent images in two channels for FAST signals and mCherry labelled bacteria. Given the good signal-to-noise ratio of mCherry images, we applied for each time point Otsu's thresholding algorithm on the entire z-stack in order to obtain a 3D segmentation of bacteria. The Otsu segmentation did not allow to isolate bacteria when they were too dense, for instance in eSLAPs, or after prolonged infection when cells were full of bacteria. Hence, we detected objects that could either be single bacteria or clusters of bacteria. We tracked each segmented object from frame to frame based on their similarities in size and location. Our method took benefit from the fact that cytosolic bacteria were moving very fast compared to those encapsulated in eSLAPs. The growth rates of bacteria inside eSLAPs were computed by fitting the dynamics of segmented mCherry volumes to an exponential function ( ) = % $& , with the growth rate and % the 585 initial volume of the vacuole. The doubling time t was then computed according to the following formula: t = !"# $ . No growth was observed for the ∆hlyA strain in long-term vacuoles tracked by the co-occurrence of FAST and mCherry signals (based on the spherical shape of the object). For the WT and the prfA* strains, an object was classified as a growing eSLAP if it met the two following criteria: (a) the initial size was equal to or larger than the size a of single bacterium (32 voxels) (S13 Fig), and (b) the size of the object at least doubled over 590 the whole track. The fraction of the objects in which Lm grew was then computed as the ratio of the number of tracked vacuoles that at least doubled their size during the 8-h course of the movie (thus matching a and b) to the initial number of objects bigger than 32 voxels on the first frame of the movie (S13 Fig), which is a good proxy for the initial number of entry events. To quantify LLO-FAST signals in a given vacuole, we used the binary mask of mCherry labelled objects to measure the average intensity in the corresponding region of the 595 FAST image. Image computing was performed using MatLab.

Quantification of YFP-CBD signals
Images were first z-projected by maximum intensity. For eSLAPs we measured YFP fluorescence intensity per pixel in ROIs with an area of 16 pixels that were manually positioned at each time point according to the mCherry signal of bacteria. For cytosolic bacteria, the mCherry images were segmented to get the masks of 600 the individual bacteria. Segmented objects were scored as cytosolic if their size was below 40 pixels. At each time point, we measured the average value of fluorescence intensity per pixel in the population of bacteria that were cytosolic. The average background YFP-CBD signals per pixel measured in the cytosol was subtracted to each value for normalization.

Immunofluorescence or LysoTracker staining of infected cells 605
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, 610 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), Acti-stain 488 phalloidin (Cytoskeleton #PHDG1, 70 nM) and DAPI (0.1 μg/μl) for 30 min. After three 615 additional washes, cover glasses were finally mounted on microscope slides with Fluoromount mounting medium (Interchim). Rabbit monoclonal primary antibodies from Cell Signalling Technologies against Rab5 (Cat #3547, RRID:AB_2300649), Rab7 (Cat# 9367, RRID:AB_1904103) and LAMP1 (Cat# 9367, RRID:AB_2687579) were used at a 1:200 dilution; rabbit polyclonal antibodies against LC3 (MBL International Cat# PM036, RRID:AB_2274121) were used at a 1:500 dilution. To quantify the number of vacuoles that associated with the indicated markers, 9 to 14 microscopic fields were examined from coverslips (or from live infection for the LysoTracker staining), and processed with Fiji. A vacuole was defined as a round aggregate of at least 4 bacteria (labelled with either mCherry in immunofluorescence experiments, or with eGFP in live experiments) that did not colocalize with actin staining.

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The presence of each marker was assessed by seeking for a corresponding fluorescent signal in the vicinity of vacuoles, and displaying a shape similar to the edge of the vacuole.

Quantification of eGFP signals as a reporter for P hlyA induction in intracellular bacteria
LoVo cells were infected as described above for immunofluorescence experiments with bacteria that constitutively expressed mCherry, as well as eGFP under the control of the PhlyA promoter. At 1 and 3 h p.i., 640 cells were fixed and stained with Acti-stain 670 phalloidin (Cytoskeleton #PHDN1-A, 70 nM) and DAPI as for immunofluorescence experiments. Fifteen to twenty fields per condition and time point were imaged with a Nikon Ti epifluorescence microscope as described above, and processed with Fiji. Images were first zprojected by maximum intensity, then bacteria were segmented using Otsu's thresholding algorithm on the mCherry signal. The objects were automatically counted and mapped on the images using the particle analyser.
To split grouped bacteria, images were processed with Watershed segmentation algorithm and both mCherry and GFP average signals were measured. For each segmented bacterium, intrabacterial eGFP and mCherry signals were quantified, and intensity of the eGFP reporter of PhlyA activity was normalized to mCherry intensities. Kruskal-Wallis non-parametric test followed by Dunn's correction for multiple comparisons was used for statistical testing between conditions. 650 equipment that was instrumental to this work, and providing expert support whenever needed. We are indebted to the IBENS animal facility and especially to Eléonore Touzalin for kindly supplying the mice blood used in haemolysis assays.

Funding
This project received support from ANR (LiVaLife-CE15-PRC-2020) for AL's and ND's groups. Work in the group of AL also received support under the program "Investissements d'Avenir" implemented by ANR

Declaration of interests
The 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.      Most Myc-tagged protein constructs were detected by immunoblotting in the corresponding bacterial pellet fraction, indicating that transgenes were expressed, even though in varying amounts (B, lanes 2, 4-7). Constructs harbouring the LLO SP or full-length LLO were recovered in bacterial supernatants (C, D, lanes 3-7), suggesting that the SP of LLO promoted Sec-dependent export of not only of FAST or FAST-tagged 920 proteins, but also of eGFP-fusion proteins. The secretion of eGFP-tagged proteins seemed less efficient than that of FAST-tagged protein (C, compare lane 3 with 4; D, compare lane 5 with 6), consistent with previous reports that eGFP is a poor substrate for Sec-dependent secretion [18]. Constructs devoid of signal peptides were not detected in supernatant fractions (C, D, lanes 1-2), arguing against the release of proteins into the culture medium due to bacterial lysis.

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For technical reasons likely due to the small size of FAST-Myc (15 kDa), it was not or barely detected by immunoblotting (B, D, lanes 1, 3); nevertheless, a strong signal corresponding to this polypeptide was visible on Coomassie-stained gels of the supernatant fractions, attesting of its secretion (C, lane 3). For bacterial pellet fractions (A, lanes 1, 3), signal from other proteins masked possible bands from that polypeptide; however, observation in microscopy (E) confirmed the non-secreted form of FAST was also produced.

S2 Fig. Fluorescent measurements of FAST-or eGFP-tagged proteins released in bacterial culture supernatants.
Six Lm strains expressing FAST-or eGFP-tagged proteins were cultured in LSM, then fluorescence intensities were measured on the filtered supernatants of each culture in presence of 5 μM HBR-3,5DM. For normalisation between FAST and eGFP signals, intensities were expressed in arbitrary units where 100 A.U. 940 corresponds, for each reporter, to the intrabacterial fluorescence emitted by a suspension of equal volume of Lm (OD600nm = 1) that expresses constitutively either non-secreted FAST or eGFP under the PHYPER promoter. Residual fluorescence measured in the culture medium of strains producing non-secreted FAST or eGFP represents bacterial lysis. All values below 1 were considered below the detection limit for this experiment, and plotted as 1 (i.e. 10 0 ). Normalized values, means and standard deviations from three independent 945 experiments were plotted. p-values represent the results of two-tailed Student's t-tests with equal variance assumption. Fig 1C (constitutive expression from a pSU2.1rp vector) was assessed by immunoblotting with anti-Myc antibodies of bacterial total extracts culture supernatant fractions, with or without induction of secretion by the T3SS using Congo red, and with or without TCA precipitation in order to concentrate samples.

S4 Fig. Exponential growth of mCherry-labelled bacteria in infected LoVo cells.
Dynamics of the total intracellular bacterial population were measured by segmentation of mCherrylabelled bacteria, in control wells recorded in parallel to the accumulation of SP-FAST in the cytoplasm (Fig  2). To get an estimate of the number of bacteria in each field, the total volume occupied by bacteria (the number 960 of voxels that were labelled with mCherry) was divided by the average size of bacteria (32 voxels). Each colour represents an independent biological replicate (in blue, two wells were recorded in the same experiment). The exponential fit associated to each growth curve is displayed as orange dashed lines.   (B) Growth curves of three Lm LL195 strains harbouring pPL2-derived vectors, at 37°C in BHI. No differences in growth rates were detected, regardless of the pPL2-derived plasmid that was integrated at the tRNA Arg locus (pAD-SP-FAST or pHpPL3-mCherry) and of the genetic modification carried out at the hlyA locus (∆hlyA or ∆hlyA::SP-FAST). Curves represent the average and standard deviation of technical triplicates, displayed in linear scale (top) or in semi-log scale (bottom).

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(C) Haemolytic properties of the Lm strains producing FAST-or eGFP-tagged LLO fusions 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. The haemolytic titre of all ∆hlyA strains was null (here, only ∆hlyA::SP-FAST was plotted). Haemolytic titres were enhanced for ∆hlyA deletion strains that had been complemented by integrative pAD plasmids harbouring hlyA fusion genes under control 980 of the constitutive PHYPER promoter. Fusion with FAST-Myc or eGFP-Myc (pAD-hlyA-FAST or -eGFP) did not affect haemolytic properties, compared to a simple fusion with Myc (pAD-hlyA). None of these strains reached the intense haemolytic properties of the prfA* strain (48.9-fold above the WT strain), for which the expression of Lm virulence genes (including hlyA) is deregulated, due to the constitutive activity of the transcriptional activator PrfA [22]. The average haemolytic titres and standard deviations from three 985 independent experiments were plotted. Two-way ANOVA on log2-transformed haemolytic titres followed by post-hoc Tukey's test was used for statistical testing between conditions. ns, non-significant; ***, p < 10 -3 , ****, p < 10 -4 .  LoVo cells were infected with Lm carrying an integrated pHpPL3-mCherry plasmid and secreting FAST-LLO due to an in-frame C-terminal fusion with FAST at the hlyA locus.
(A) Number of bacteria inside eSLAPs over time. mCherry signals allowed the segmentation of bacteria and their counting. The exponential fit associated to each growth curve is displayed as orange dashed lines. The black horizontal dashed line represents the volume of one average doubling event since the first frame.  did not rupture over the same time-course. Negative values correspond to signals below cytosolic levels, indicating that YFP-CBD was excluded from eSLAPs when they formed. In contrast, when bacteria were exposed to the host cytosol, they became positively stained. Error bars indicate the standard error of the mean.  The first bin of the distribution (left of the green dotted line) corresponds to objects smaller than the size of bacteria that were discarded when counting the number of entry events. The bins between the green dotted and orange dashed lines correspond to single bacteria, the size of which was in the range of 32 to 64 voxels. The orange dashed line marks the limit between objects that correspond to individual bacteria (left) and clusters of bacteria (right). The mean of the distribution (n = 354 objects from 15 pooled experiments) was equal to 75 S5 Movie. 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. Cyan, CBD-YFP channel; Orange, mCherry channel. The cell outline is shown with a purple dashed line. Scale bar, 5 μm.