Listeriolysin O promotes the intravacuolar growth of Listeria monocytogenes in epithelial cells

15 Upon entry into host cells, Listeria monocytogenes (Lm) was described to escape rapidly from internalisation vacuoles and proliferate only after gaining access to the cytosol. Vacuole escape depends upon three secreted virulence factors: the pore-forming toxin listeriolysin O (LLO) and two phospholipases. To quantify the dynamics of vacuolar escape, we used FAST fluorescent tags to monitor bacterial secretion into enclosed compartments. By tracking fluorescently-labelled vacuoles, we quantified the heterogeneity of 20 Lm residence time in primary vacuoles formed in epithelial LoVo cells. Although half of the bacterial population escaped from vacuoles within 13 minutes after internalisation, a fraction of it remained entrapped several hours in Long Residence Vacuoles (LRV), for both wild type and LLO-deficient strains. Unexpectedly, Lm replicated inside LRVs at a rate similar to that in the cytosol. LRVs were decorated with LLO-FAST and LLO was necessary for bacterial proliferation in these compartments, suggesting that 25 permeation of vacuolar membranes sustained growth. LRVs displayed similarities with the spacious Listeriacontaining phagosomes described in macrophages, and could constitute an alternative replication niche for Lm in epithelial cells. . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for . http://dx.doi.org/10.1101/2019.12.23.886689 doi: bioRxiv preprint first posted online Dec. 23, 2019;


Introduction
Bacterial pathogens harness distinct strategies to colonize their host and take advantage of its resources, for instance by adopting an intracellular lifestyle. Internalisation into host cells can allow invasive bacteria to cross the organism barriers, escape humoral immune surveillance, or disseminate throughout the organism as cargo of circulating cells. After internalisation, bacteria are entrapped inside primary vacuoles from where 5 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 (reviewed in Salcedo & Holden, 2005;Di Russo Case & Samuel, 2016). Others, such as Shigella flexneri or Listeria monocytogenes, typically do not grow inside 10 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 & Lebreton, 2014;15 Radoshevich et al, 2015). 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 & Cossart, 2018). All 20 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 & 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 25 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 30 activity of the phospholipases PlcA and PlcB and the influence of host factors render LLO dispensable for vacuolar escape in several human epithelial cell lines (Marquis et al, 1995;Burrack et al, 2009). 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 slowly therein, with a doubling time in the range of 8 h (Birmingham et al, 2008). 3 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 in the primary entry vacuoles together with invading bacteria, the fluorescent dye stained the vacuolar space until it broke down.
Alternative strategies were based on the assessment of vacuole rupture events and bacterial access to the host 5 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 (Pizarro-Cerda & Cossart, 2018;Henry et al, 2006).
Cytoplasmic FRET probes that are cleaved by a β-lactamase secreted by invasive bacteria have also been 10 shown to be efficient reporters of vacuole rupture (Ray et al, 2010;Quereda et al, 2015). Even though these techniques 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 these tools has been used so far to determine the distribution of Lm residence time in entry vacuoles, which is limiting the interpretation of variations found between conditions. 15 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 are internalised into cells, and the moment when the vacuole membrane is ruptured. We devised a strategy relying on the tagging of proteins secreted by bacteria with the FAST reporter system (Plamont et al, 2016). 20 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 make this system an ideal candidate for tagging secreted proteins and imaging them in real time. 25 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 could last for several hours and are 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 efficiently in these 30 compartments, suggesting that besides its role in vacuolar escape, LLO could contribute to set up an intravacuolar niche prone to Lm replication in epithelial cells. 4

FAST-tagging of secreted bacterial effectors for live microscopy
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. In order to investigate the ability of Lm to secrete 5 FAST-tagged proteins via the Sec pathway, a set of integrative plasmids harbouring gene fusions under control of the P HYPER promoter were designed (Fig EV1A, Appendix Fig S1A). These plasmids drove the constitutive production of either FAST or eGFP, either for intrabacterial localisation, or fused in their Nterminus 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. All 10 constructs additionally encompassed a Myc tag in their C-terminus in order to allow detection by immunoblotting. Protein production and secretion by each one of these seven strains was assessed by colloidal Coomassie staining and immunoblotting against the Myc tag, on bacterial total extracts and culture supernatant fractions from overnight-grown cultures in BHI, separated by SDS-PAGE ( Fig EV2). All transgenes were efficiently expressed by Lm strain LL195, even though in varying amounts. As expected, 15 constructs harbouring either the LLO SP, or full-length LLO, were recovered in bacterial culture supernatants, suggesting 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 (Fig EV2C, 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 20 secretion pathway.
To assess whether the FAST reporter system remained fluorescent after secretion, we quantified the fluorescence signals in the supernatants of bacterial cultures grown overnight in iLSM ( Fig 1A). In presence of 5 μM of HBR-3,5-DM, fluorescence was detected in the supernatants of strains secreting SP-FAST or LLO-FAST. Fluorescence intensities in the culture medium of strains producing non-secreted FAST or eGFP 25 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 retain their fluorescent properties after undergoing secretion trough the bacterial Sec system. By calibrating fluorescence measurements with a standard curve of known FAST:HBR-3,5-DM concentrations diluted in the same minimal medium, we estimated the secreted concentration of tagged proteins. The concentration of 30 secreted SP-FAST after an overnight culture was 325±55 nM, and that of LLO-FAST around 28±6 nM.
Several attempts by others with various Gram-negative or -positive bacteria (Dammeyer & Tinnefeld, 2012;van der Ploeg et al, 2012), and our own unpublished observations using tagged Lm virulence factors, indicated 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 35 5 rise to fluorescent signals in culture supernatants, even though in a range 10 fold lower than that obtained for the secretion of SP-FAST. A consistent proportion of eGFP undergoing Sec-dependent secretion is thus able to acquire its mature fold in bacterial culture medium, at least in iLSM, and when not fused to a bacterial effector.
To evaluate the versatility of FAST as a reporter of bacterial secretion, we next asked if FAST was 5 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 substrates, we tagged C-terminally with FAST-Myc the effectors OspF and IpaB (Fig EV1B), 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-10 III dependent secretion by addition of Congo red. By immunoblotting these fractions against the Myc-tag, we observed that tagged OspF and IpaB were secreted into the bacterial culture medium upon Congo red induction ( Fig EV3A). Constitutive secretion of both tagged effectors was observed when using a ∆ipaD mutant strain for which translocation lacks gating controls (Ménard et al, 1994) (Fig EV3B). We then assessed whether the fusion proteins secreted by the ∆ipaD strain had retained their fluorescent properties, 15 by measuring fluorescence intensities in the supernatants of bacterial cultures grown overnight in M9 medium ( Fig 1B). 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 20 secretion.
We then investigated whether the FAST reporter system was suited for intracellular detection in realtime microscopy of proteins secreted during infection. 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, Movie EV1). FAST fluorescence increased uniformly over time in the cytoplasm of infected cells (Fig 2A). 25 At 562 nm-the wavelength specific for FAST:HBR-3,5-DM-, fluorescent signals accumulated in cells infected with a strain producing SP-FAST, and not in a control isogenic strain that constitutively expressed mCherry ( Fig 2B). In infected cells, fluorescence intensity-which corresponds to intracellular concentration of SP-FAST-increased exponentially over time (Fig 2C), likely mirroring the exponential growth of Lm in the host cytosol. After an exponential increase, the intracellular fluorescence dropped suddenly, 30 corresponding to the death of infected cells and the permeation of their membranes. The distribution of exponential fluorescence increase rates in FAST fluorescence was indicative of the variability in the infection rates among infected cells (Fig 2D). The median rate was 0.66 h -1 , corresponding to a doubling time of 63 min. Consistently, a median bacterial growth rate of 0.72 h -1 (doubling time of 58 min) was measured in similar conditions of infection and illumination by segmenting mCherry-labelled bacteria, and then 35 measuring intrabacterial mCherry signals over time. The long tail of the distribution of exponential 6 fluorescence increase rates likely reflected additional entries due to cell-to-cell spread from neighbouring cells. 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 5 signals were diluted and therefore only became visible after several hours of infection, once they had accumulated sufficiently enough to be significantly discriminated from non-specific signals. Meanwhile, we reasoned that if Lm was confined in micron-sized internalisation vacuoles, the higher concentration of secreted FAST molecules in a reduced volume could allow their detection and tracking until the rupture of vacuole membranes, thereby providing an accurate measurement of the lifetime of primary vacuoles (Fig   10   3A). Indeed, we observed that SP-FAST signals were enhanced in micro-sized compartments that colocalized with mCherry-expressing bacteria within minutes after bacterial adhesion to cells, until these signals suddenly dropped when vacuoles ruptured ( Fig EV1C). The median value for the residence time of the WT strain was of 12.7±0.7 min ( Fig 3E). 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 20 LLO in epithelial cells (Marquis et al, 1995;Burrack et al, 2009). 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 number of intact vacuoles were still observed 3 h p.i. (4.6 % for the WT strain; 6.2 % for the ∆hlyA mutant) ( Fig 3D). The fact that the WT strain remained entrapped in Long Residence Vacuoles (LRVs) in nearly identical proportions as the ∆hlyA strain could either suggest that a sub-population of WT Lm failed to 25 escape primary vacuoles in spite of LLO secretion, or that LLO was not produced in this sub-population 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.

Role of LLO for the long-term vacuolar residence and intravacuolar replication of Listeria
To examine whether LLO was produced and secreted by bacteria that remained entrapped in LRVs, we 30 engineered a Lm strain where LLO was C-terminally fused with FAST at the endogenous hlyA locus ( Fig   EV1C). 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 15kDa FAST-Myc tag should not interfere with the function of the protein it 7 reports for. We thus assessed the haemolytic properties of the strain expressing hlyA-FAST, which did not differ from that of the WT strain (Appendix Fig S2). 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 was also constitutively expressing mCherry, which allowed us to segment and track bacteria in 3D during infection. When imaging mCherry-labelled bacteria and LLO-5 FAST from 2 h post-infection (p.i.) in LoVo cells, we observed that Lm could remained entrapped inside LRVs for 9 h before the rounded structure of LLO-labelled membranes eventually disrupted and bacteria dispersed into the cytosol (Fig 4A, Movie EV4). Strikingly, the volume occupied by the mCherry signal increased with 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 By tracking growing vacuole and measuring their volume over time, we determined the growth rate of Lm inside long-lived vacuoles. Lm grew exponentially in LRVs, with a rate similar to that of free bacteria in the cytosol (Fig 4B, Fig EV5A). The observed doubling time (90 min in this experimental setup; or down to 1h when the intensity and frequency of illumination were reduced as in Fig. 2D) was consistently shorter than that previously described in SLAPS, which was in the range of 8 h (Birmingham et al, 2008). 20 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 to assess the variability in LLO abundance in these compartments. LLO-FAST signals measured in LRVs were greater than background levels but displayed a broad range of dynamics, indicating that LRV formation and 25 maintenance was independent of the amounts of secreted LLO (Fig EV5B). In some LRVs, LLO-FAST accumulated linearly over time, while others displayed large-scale fluctuations in the signal. Some LRVs yielded a strong signal while others displayed low levels of decoration by LLO. The lifetime of LRV was correlated with neither the average concentration of LLO (Appendix Fig S3A) nor its maximal level (Appendix Fig S3B), suggesting that LLO concentration poorly influenced the probability of Lm escape from 30 these structures. Consistently, we observed that not only WT and ∆hlyA Lm could reside in long-term vacuoles ( Fig 3D, Fig 4C-D), but that LRVs were observed even 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 35 we used was 10 5 -fold higher than that of the WT strain (Appendix Fig S2). Highly elevated levels of LLO 8 thus did not impede the ability of Lm to reside and multiply inside LRVs for several hours. This feature is contrasting with the properties of SLAPs previously described in phagocytes, which required moderate amounts of LLO secretion for their formation (Birmingham et al, 2008). Nevertheless, LLO hyperproduction facilitated bacterial escape from LRVs to a certain extent. Indeed, we observed in live-cell imaging (Fig 4C-D) that the very high levels of LLO secretion of the prfA* strain hastened Lm escape from LRVs. In 5 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 ( Fig 4E).
Although the presence of LLO was not required for LRV formation and Lm escape, we observed that the ∆hlyA Lm strain was unable to proliferate inside LRVs ( Fig 4C). Individual tracking of LRVs revealed that ∆hlyA bacteria barely grew when no LLO was secreted (Fig 4D). Similarly with SLAPs, LRVs thus required 10 that bacteria secreted LLO to allow intravacuolar growth. The quantity of secreted LLO did not appear to affect bacterial growth, since the prfA* strain replicated with a similar rate as the WT strain ( Fig 4D).
Consistently, the growth rate of LLO-FAST-secreting bacteria in LRVs was correlated with neither the average concentration (Appendix Fig S3C) nor the maximal level of LLO secretion (Appendix Fig S3D). 15 The LRVs in which Lm replicated (Fig 4) likely originated from internalisation vacuoles from which bacteria had failed to escape ( Fig 3D). However, one cannot exclude that the vacuoles where Lm were found to replicate after 2 h p.i. might instead derive 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 if the LRVs where Lm proliferated were actually primary vacuoles, we monitored by confocal 20 spinning disk fluorescence microscopy 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 cytoplasm, because the cell wall-binding domain (CBD) from the Lm phage endolysin Ply118 binds the cell wall of Lm with high affinity. Bacteria that replicated within LRVs remained unlabelled with YFP-CBD, 25 until the vacuole ruptured and bacteria dispersed throughout the cytosol (Fig 5A, Movie EV6). This result rules out the possibility that bacteria underwent canonical autophagy or cell-to-cell spread after a first exposure to the host cell cytoplasm, and thereby confirms that LRVs where Lm replicates derive from internalisation vacuoles.

Origin and properties of Listeria long residence vacuoles in epithelial cells
Because the replication compartments we observed were reminiscent of the SLAPs, we hypothesized that 30 they could originate from a process analogous to LC3-accosiated phagocytosis (LAP), except it would occur in epithelial cells rather than in phagocytes. We thus endeavoured to better 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 35 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.
this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for . http://dx.doi.org/10.1101/2019.12.23.886689 doi: bioRxiv preprint first posted online Dec. 23, 2019; 9 early endosomal marker Rab5, while they were positive for the late endosomal marker Rab7, the lysosomal marker LAMP1, as well as LC3 ( Fig 5B). These are typical markers of SLAPs, suggesting that, similar to what occurs in phagocytes, LC3 could be lipidated and the noncanonical autophagy machinery recruited to the entry vacuole in epithelial cells. In spite of the presence of the lysosomal marker LAMP1, the pH inside the LRVs remained neutral, as revealed by their absence of staining when using the acidophilic fluorescent 5 probe LysoTracker Deep Red ( Fig 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.

10
Addressing 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 brought evidence that FAST offered a versatile, convenient opportunity for tackling this challenge. We took advantage of it to measure the lifetime of Lm internalisation vacuoles, and to monitor the vacuolar localisation of the secreted Lm virulence factor LLO in live cells.

Real-time imaging of LLO during infection
On fixed samples, addressing 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, has been addressed by electron microscopy using immunogold labelling (Quinn et al, 20 1993;Coelho et al, 2018). However, the precise dynamics of infectious processes are not addressed with fixed-cell studies. In addition, the high special resolution gained by electron microscopy limits the overview of events at a cellular scale. As a complementary approach, LLO-eGFP fusions that were ectopicallyexpressed in host cells enabled live imaging studies, which yielded precious insight into the dynamics of LLO localisation at membranes and turnover (Chen et al, 2018). Nevertheless, such studies remained limited 25 by expression from a transfected plasmid, which could not reproduce the concentrations, precise localisation and orientation of the insertion of LLO oligomers into vacuole membranes that are obtained with endogenous bacterial secretion. In addition, in non-infected cells, host cell signalling pathways and membrane dynamics do not undergo the same perturbations as in infected cells. Here, we report (a) that the FAST system can be used to tag LLO without loss of function, (b) that the LLO-FAST fusion can be 30 expressed under its endogenous promoter and secreted by Lm in infected cells, (c) that the vacuoles it decorates can be imaged with accuracy, and (d) that some of these vacuoles unexpectedly last for several hours. this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for .

LRVs, an alternative replication niche for Listeria monocytogenes
We observed that in LoVo epithelial cells, a consistent proportion of Lm failed to escape from the internalisation vacuole, but instead remained and replicated efficiently inside LRVs, which were positively labelled by LLO-FAST. The decoration of these compartments by LC3, Rab7 and LAMP1 as well as their neutral pH were reminiscent of the SLAPs previously described in phagocytes (Birmingham et al, 2008), and 5 which derive from LAP (Mitchell et al, 2018). To our knowledge the LAP of Lm has not been previously reported to occur in epithelial cells, in contrast with Sf that can take advantage of a process similar to LAP Whereas the LRVs that we observed in LoVo cells displayed similarities with SLAPs, they were notably 20 distinct from LisCVs, an intravacuolar persistence niche of Lm recently described in human hepatocytes and trophoblast cells (Kortebi et al, 2017). Indeed, contrary to LRVs and SLAPs, LisCVs did nor derive from primary vacuoles. Instead, they were described to form later during the intracellular cycle of Lm, by entrapment within vacuoles of bacteria having lost ActA-dependent motility. In line with this, bacteria found in LisCVs were labelled with YFP-CBD, while the bacteria we observed in LRVs were not. ActA function 25 was indifferent to the formation of LRVs, since they were also observed when using a prfA* strain, where actA is constitutively expressed (Reniere et al, 2015). Moreover, whereas LRVs were lipidated by LC3, LisCVs were LC3 negative. Last, the bacteria residing within LisCVs appeared to be in a viable, nonculturable state, while Lm replicated in LRVs. Altogether, though occurring in epithelial cells, the features we describe for LRVs are consistent with compartments similar to SLAPs, and distinct from LisCVs. this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for . http://dx.doi.org/10.1101/2019.12.23.886689 doi: bioRxiv preprint first posted online Dec. 23, 2019; 11 anticipate that the excessive production of LLO by this strain would quickly destabilise vacuolar membranes (Ripio et al, 1997). In line with this view, in phagocytes Lm residence in SLAPs was favoured when using strains producing moderate amounts of LLO (Birmingham et al, 2008). One possible explanation for this difference would be if active repair mechanisms were at play in LoVo cells, bringing additional membrane components to the growing compartments. Whether the recruitment of lipidated LC3 and noncanonical 5 autophagy is involved in this process might be a track for future investigations. As an alternative, rapid scavenging of LLO, made possible by its PEST sequence (Chen et al, 2018), could participate in maintaining a balance between vacuole permeation and rupture.
The relevance of LRVs in the context of infections remains to be explored. One possible aspect to be examined in future studies is to which extent this additional replicative niche contributes to the survival of 10 Lm within epithelial cells and tissues, not only in LoVo epithelial cells but also in possible in vivo niches, and to the maintenance of an equilibrium between bacterial fitness, host responses and host-inflicted damage.
Innate immune sensing of the pathogen would likely differ when Lm is thus entrapped, which could modulate the bacterial-host interplay. Vacuolar bacteria that cannot spread from cell to cell might also constitute chronic forms of infections by dampening immediate damage to the host. Together with LisCVs, 15 with SLAPs, and with growth-restricting-processes associated with xenophagy (Mitchell et al, 2018), the LRVs we describe here in LoVo cells thus add-up to the notion that a variety of Lm intravacuolar forms may coexist in infected cells, each contributing to the complexity in sorting out possible outcomes of the infectious process. 20 Beyond the progress allowed for live detection of LLO and the description of this replicative compartment, the FAST reporter appears as a promising tool for live imaging studies of bacterial secretions adapted to a broad range of bacterial models and secretion systems. As a possible application, we have shown here that the simple accumulation of secreted FAST into Lm internalisation vacuoles allowed single particle tracking of these compartments, from the moment of their formation to the moment of their rupture, this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for . 12 through the Sec pathway (Dinh & Bernhardt, 2011;Peters et al, 2011;Dammeyer & Tinnefeld, 2012;Khatib et al, 2016), 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 (Enninga et al, 2005;Gawthorne et al, 2016).

FAST, a versatile fluorescent reporter of bacterial secretion
Nevertheless, the toxicity in eukaryotic cells of the biarsenite dye used for FlAsH labelling and the rather 5 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); this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in Lm. Whenever required, media were supplemented with antibiotics for plasmid selection (chloramphenicol, 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 (gfAL001, Appendix Fig S1A) or Sf (gfAL002, Appendix Fig S1B) using 5 the online Optimizer application (http://genomes.urv.es/OPTIMIZER/) in guided random mode. The optimized sequences were obtained as synthetic Gene Fragments (Eurofins genomics). gfAL001 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 pPL2 backbone (Fig EV1), gfAL001 was amplified with primers 10 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 UTR hlyA -SP-FAST-Myc amplicon was inserted instead of UTR hlyA -eGFP into the EagI-SalI restriction sites of pAD-cGFP, thus generating pAD-SP-FAST, where FAST is under control of the P HYPER constitutive promoter (Fig EV1A). pAD-FAST, pAD-eGFP, pAD-SP-15 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 (Fig EV1A).
After cloning in E. coli NEB5α, 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 (Fig EV1C), pMAD-∆hlyA::FAST was created by amplifying For Sf constructs, ipaB and ospF were amplified from M90T genomic DNA with primers oAL703-4 and 707-8 respectively, and gfAL002 was amplified with primers oAL705-6. pSU2.1-OspF-FAST (Fig EV1B) was obtained by inserting an oAL707-6 amplicon overlapping ospF and FAST-Myc, with a BamHI restriction linker, in place of mCherry into the KpnI-XbaI restriction sites of pSU2.1rp-mCherry (Campbell-35 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in Valois et al, 2015). pSU2.1-IpaB-FAST was generated by replacing ospF with ipaB (oAL703-4) at the KpnI-BamHI sites (Fig EV1B). After cloning in E. coli NEB5α, these plasmids were introduced in Sf M90T by electroporation.
The complete lists of bacterial strains and oligonucleotides used in this work are supplied as Appendix Tables S1 and S2, respectively. 5

Bacterial total extracts or secreted protein analysis
Bacterial total extracts or culture supernatants were recovered from 1 ml of Lm strains grown to a OD 600nm 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 a 10 OD 600nm 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 OD 600nm of 40, and incubated at 37°C for 45 min. Bacteria were eliminated 15 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 20 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 two-fold 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% 30 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 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in

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, 180 rpm. Likewise for secretion by Sf, a culture in TSB was diluted to 1:10 th in M9 medium supplemented 5 with 0.2% glucose and 10 μg/ml nicotic acid. From 1 ml of culture, bacterial pellets were collected by centrifugation of the cultures at 6,000 × g, then washed in PBS and resuspended in 1 ml of PBS. The culture supernatants were filtered (0. Each experiment was reproduced three times independently. Lm strains were grown in BHI medium until they reached early stationary phase (OD 600 of 2 to 3), washed . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for . http://dx.doi.org/10.1101/2019.12.23.886689 doi: bioRxiv preprint first posted online Dec. 23, 2019; 16 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 5 μg/ml in order to kill extracellular bacteria.

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  this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for . http://dx.doi.org/10.1101/2019.12.23.886689 doi: bioRxiv preprint first posted online Dec. 23, 2019;

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 5 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 10 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 pre-coated 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, 15 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 this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for . http://dx.doi.org/10.1101/2019.12.23.886689 doi: bioRxiv preprint first posted online Dec. 23, 2019 18 were acquired with Nikon apochromat 60x objective lenses (NA 1.4). Image acquisition and microscope control were actuated with the μManager software, and processed with Fiji. Each picture is representative of the infected cell population.

Acknowledgements
We are grateful to Marie-Aude Plamont, Vinko Besic, Sebastian Rupp and Alison Tebo for their 5 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 renewed proofs of 10 enthusiasm and insightful discussion.

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. this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for . this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for . http://dx.doi.org/10.1101http://dx.doi.org/10. /2019  this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for . http://dx.doi.org/10.1101/2019.12.23.886689 doi: bioRxiv preprint first posted online Dec. 23, 2019;  this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for . http://dx.doi.org/10.1101/2019.12.23.886689 doi: bioRxiv preprint first posted online Dec. 23, 2019 27 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.

Figures
this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for . http://dx.doi.org/10.1101/2019.12.23.886689 doi: bioRxiv preprint first posted online Dec. 23, 2019; this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for . http://dx.doi.org/10.1101http://dx.doi.org/10. /2019  this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in for 3 h with mCherry-expressing bacteria (in red). For acidity staining, LoVo cells infected for 2 h with eGFP-expressing bacteria (in red) were stained with LysoTracker Deep Red (in green), and observed 1 h afterwards. Observations were performed on an inverted spinning disk microscope. Scale bars, 5 μm.
. CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.
this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for . http://dx.doi.org/10.1101/2019.12.23.886689 doi: bioRxiv preprint first posted online Dec. 23, 2019;

Movie EV3. Background signals in the FAST channel when cells were infected with bacteria
10 that do not secrete FAST.
LoVo cells infected with Lm expressing mCherry were observed between 0 and 3.25 h post-infection by spinning-disk microscopy. Acquisition parameters and contrasts were sets as in Mov. S2. Green, FAST channel (non-specific signals); red, mCherry channel; blue, SiR-actin channel. Scale bar, 10 μm.  this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in Epifluorescence microscopy observation of strains producing non-secreted FAST or eGFP. Scale bar, 2 μm.

Movie EV4. Observation of the decoration of LRVs by LLO-FAST in Listeria cells infected by
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 10 3-7), suggesting that the SP of LLO promoted Sec-dependent export of not only of FAST or FAST-tagged 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 . CC-BY-NC-ND 4.0 International license It is made available under a perpetuity.
this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in reports that eGFP is a poor substrate for Sec-dependent secretion (Dammeyer & Tinnefeld, 2012). 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.
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 5 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.
. CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for . http://dx.doi.org/10.1101/2019.12.23.886689 doi: bioRxiv preprint first posted online Dec. 23, 2019;

Table of contents of the Appendix
Appendix figures S1 to S4 Appendix tables S1 to S2 5 this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in 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 5 ∆hlyA deletion strains that had been complemented by hlyA fusion genes under control of the strong, constitutive P HYPER 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 10 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.

Appendix figures
. CC-BY-NC-ND 4.0 International license It is made available under a perpetuity. this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in this preprint (which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for . http://dx.doi.org/10.1101/2019.12.23.886689 doi: bioRxiv preprint first posted online Dec. 23, 2019;