S.Tm invasion of epithelial cell lines and the murine gut absorptive epithelium differentially requires SPI-4 and the TTSS-1 effectors SopB, SopE, SopE2, and SipA

Efficient S.Tm invasion of epithelial cell lines requires TTSS-1 function [46]. In addition, invasion of polarized MDCK cells involves the SiiA-E adhesin system encoded on SPI-4, which is dispensable for entry into non-polarized cell lines (S1A–S1C Fig) [47]. In S.Tm^wt (SL1344), the TTSS-1 effectors SopB, SopE, and SopE2 have been shown to predominantly drive ruffle-mediated entry, while the actin-binding effector SipA has a less prominent role [8,9,17,27]. We used automated microscopy to explore the generality of these findings in non-polarized epithelial cell lines of human (HeLa), canine (sub-confluent MDCK), and murine origin (m-ICc12). Joint deletion of the ruffle-inducers (i.e. ΔsopBEE2) essentially abolished S.Tm invasive behavior in all cases, similar to deletion of all four effectors (i.e. deletion of sopBEE2sipA;“Δ4”) (Fig 1A). By contrast, we were unable to detect a significant invasion defect upon deletion of SipA (ΔsipA) in either cell line (Fig 1A). In fact, at high pathogen densities, S.Tm^ΔsipA invaded HeLa and m-ICc12 marginally better than S.Tm^wt. These data, supported by bacterial plating assays (S1D Fig), validate the ruffle-inducers SopBEE2 as the key drivers of S.Tm invasion into diverse non-polarized epithelial cell lines.

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Fig 1. S.Tm invasion of cultured epithelial cell lines and the murine gut absorptive epithelium differentially depends on bacterial virulence factors.

(A) SopBEE2 drive S.Tm invasion of non-polarized epithelial cell lines. Invasion efficiency of S.Tm/pssaG-GFP reporter strains in the indicated subconfluent epithelial cell lines, infected for 20min over a range of MOIs, and analyzed at 4h p.i. by automated microscopy. Data points correspond to mean +/- range of two to three replicate infections. (B-D) SipA drives S.Tm gut absorptive epithelial cell invasion in vivo in mice. Sm-pretreated C57BL/6 wild-type mice were orally infected with the indicated S.Tm/pssaG-GFP reporter strains for 12h. The three strains to the right of the dashed line are also deficient for TTSS-2. (B) S.Tm CFU counts in cecum content. (C) Representative micrographs of the cecal mucosa upon infection with the indicated strains. Lu.—Lumen; Ep.—Epithelium; L.p.—Lamina propria. White arrow heads indicate intraepithelial S.Tm. Scale bar: 10μm. (D) Quantification of intraepithelial S.Tm per 20μm section. In B and D each data point corresponds to one animal. Line at median. Kruskal-Wallis with Dunn’s post-test (n.s., not significant; *p<0.05; ***p<0.001). (E-F) SipA cannot promote epithelial cell invasion when present on a co-infecting bacterium. Wild-type mice were infected with a 1:1 mix of the indicated S.Tm/pssaG-GFP-reporter strain (rep.) and non-fluorescent helper strain (help.) for 12h. (E) S.Tm CFU counts in cecum content. (F) Quantification of intraepithelial S.Tm per 20μm section. Each data point in E-F corresponds to one animal. Line at median. Mann-Whitney U-test (n.s., not significant).

https://doi.org/10.1371/journal.ppat.1008503.g001

Due to its well-defined colonization kinetics, we chose the streptomycin mouse model of Salmonella gut infection to study S.Tm invasion of the intestinal epithelium in vivo. In this model, luminal colonization and epithelial cell invasion begins in the cecum [34]. We investigated if the first wave of invasion shows a similar timing throughout this entire gut segment. C57BL/6 wild-type mice were infected (by oral gavage; 5x10⁷ CFU) with S.Tm^wt harboring a pssaG-GFP reporter (turns GFP+ subsequent to host cell entry [36]). Microscopy scoring of tissue-lodged S.Tm-GFP+ revealed equivalent numbers of intracellular bacteria across the entire cecal length (S1E and S1F Fig). As expected, the vast majority of S.Tm invasion foci (~95%) localized to EpCam-positive absorptive epithelial cells (S1G and S1H Fig). Hence, by microscopy of the middle part of the cecum, we can sensitively quantify S.Tm invasion of gut absorptive epithelial cells in vivo. As a baseline, we infected wild-type mice for 8-12h with S.Tm^wt or isogenic strains deficient in TTSS-1 (ΔinvG) or the Sii adhesin system (Δspi-4), all harboring the pssaG-GFP reporter. S.Tm^wt were as expected abundant in the cecal epithelium, whereas the S.Tm^ΔinvG strain exhibited virtually undetectable numbers of invasion events (~200-fold attenuated at 8h; >1000-fold attenuated at 12h p.i.; S1J–S1L Fig). Deletion of spi-4 resulted in ~20-fold lower levels of tissue-residing bacteria at 8h, but only a minor difference at 12h p.i. (S1J–S1L Fig). Consequently, early invasion of the naïve murine gut absorptive epithelium critically relies on TTSS-1 and is further enhanced by the SPI-4-encoded adhesin system.

Next, we infected wild-type mice (orally as above) with S.Tm^wt, S.Tm^ΔsipA, S.Tm^ΔsopBEE2 and S.Tm^Δ4 strains to analyze the dependence on TTSS-1 effectors. All strains equally colonized the gut lumen at 12h p.i. (Fig 1B). Remarkably, and in sharp contrast to results from the cell lines (Fig 1A), the S.Tm^ΔsipA strain reached dramatically lower pathogen densities in the gut epithelium than S.Tm^wt (~200-fold lower; Fig 1C and 1D). S.Tm^ΔsopBEE2 was also attenuated, but still ~10-20-fold more invasive than S.Tm^ΔsipA (Fig 1D). In fact, the epithelial loads of S.Tm^ΔsipA were similar to those of the strain lacking all four effectors (S.Tm^Δ4; Fig 1D), or the TTSS-1 apparatus itself (S.Tm^ΔinvG; S1L Fig). Plasmid complementation restored SipA protein expression and epithelial invasion (Fig 1D and S1M Fig). Finally, similar observations were also made in a strain background lacking a functional TTSS-2 apparatus, which is important for intracellular S.Tm life (Fig 1D, right panels). Taken together, our data reveal a fundamentally different impact of TTSS-1 effectors on S.Tm invasion into epithelial cell lines—primarily driven by the ruffle-inducers SopBEE2 (Fig 1A), vs the murine gut absorptive epithelium in vivo—primarily driven by SipA (Fig 1B–1D).

SipA also affects S.Tm-induced gut inflammation (S1N Fig). Based on prior proposals [48], it remained conceivable that SipA could indirectly facilitate bacterial access to the epithelium in vivo, through inflammation-induced thwarting of protective barriers (i.e. affecting immune cell influx, mucus structure, or antimicrobial peptide production). If that was the case, then co-infection with a separate SipA-expressing strain should help S.Tm^ΔsipA achieve wild-type invasion efficiency. However, while a co-administered SipA-proficient helper strain (S.Tm^ΔsopBEE2) augmented the early signs of inflammation (median pathoscore increased from 1 to 5; S1O Fig), it did not increase the invasion efficiency of a S.Tm^ΔsipA reporter strain (Fig 1E and 1F; compare with SipA plasmid complementation in 1D). We conclude that SipA needs to be expressed by the invading bacterium itself to drive epithelial cell entry in vivo.

One feature that distinguishes the gut absorptive epithelium from common cell lines is its confluent, columnar, polarized cell arrangement. Epithelial cell polarization may be influenced by and/or affect the impact of S.Tm effectors [49,50]. We investigated if this property could account for the exquisite dependence on SipA for S.Tm invasion of the gut epithelium. MDCK cells were cultured in parallel as subconfluent non-polarized vs confluent polarized cell layers on cell culture plastic (S2A Fig). S.Tm^ΔsipA showed equal invasion efficiency as S.Tm^wt into non-polarized MDCK cells, but a ~2-3-fold attenuation in polarized MDCK cells (S2B and S2C Fig). To assess if this observation could be generalized, we cultured mouse m-ICc12 cells and human Caco-2 C2Bbe1 cells atop Transwell inserts. In agreement with previous work [47,51], m-ICc12 cells only formed a semi-polarized/tight monolayer (judged by trans-epithelial electrical resistance; TEER<50 Ωcm2) (S2D Fig), while Caco-2 C2Bbe1 cells formed a tightly polarized monolayer (TEER>1000Ωcm2; S2E Fig). SipA was dispensable for S.Tm invasion also of semi-polarized m-ICc12 cells (S2F Fig). During short-term infection of polarized Caco-2 C2Bbe1 cells, the invasion capacity of S.Tm^ΔsipA was however reduced ~2-fold compared to S.Tm^wt (S2G Fig). Hence, SipA may moderately boost S.Tm invasion of polarized epithelial cells in culture (S2 Fig), but this feature cannot by itself account for the ~200-fold impact of the SipA effector on epithelial cell invasion in the mouse gut (Fig 1C and 1D).

In summary, S.Tm invasion of the murine gut absorptive epithelium is facilitated by the SPI-4 adhesin system and critically depends on TTSS-1 delivery of the primordial effector SipA, with a surprisingly small contribution of the three ruffle-inducers SopBEE2.

Experiments in inflammasome-deficient mice verify the importance of SipA for gut absorptive epithelium invasion

Several TTSS-1 effectors, and in particular SipA, have been shown to shape the intracellular S.Tm niche and/or promote the bacterium’s replicative potential subsequent to host cell entry [45,52–54]. This could potentially skew the results above that rely on the pssaG-GFP reporter to quantify invasion efficiency in vivo. Moreover, infected epithelial cells in wild-type mice are frequently and quickly expelled into the lumen by an inflammasome response [30,55,56]. This reduces pathogen loads in the mucosal tissue and might hamper precise quantification of epithelial cell invasion efficiencies. To substantiate the observations above, we conducted S.Tm infections in inflammasome-deficient (Nlrc4^-/-) mice [57]. This allowed infections for a longer time period (18h) without overt epithelium destruction and resulted in ~50-100-fold higher total bacterial numbers in the cecal absorptive epithelium (Fig 2A–2D and S1I Fig). At these high loads, robust tissue plating experiments could be performed without problems arising from carry-over contamination by the dense luminal S.Tm population (approximately 10⁹ cfu/g content; Figs 1B and 2A).

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Fig 2. Infections in inflammasome-deficient mice confirm the dependence on SipA for S.Tm absorptive epithelial cell invasion in vivo.

(A-E) Deletion of SipA has a profound impact on absorptive epithelial cell invasion in the mouse gut. Inflammasome-deficient (Nlrc4^-/-) mice were orally infected with the indicated S.Tm/pssaG-GFP reporter strains for 18h. (A) S.Tm CFU counts in cecum content. (B) Representative micrographs of the cecal mucosa upon infection with the indicated strains. Lu.—Lumen; Ep.—Epithelium; L.p.—Lamina propria. White arrow heads indicate intraepithelial S.Tm. Scale bar: 10μm. (C) Microscopy-based quantification of intraepithelial S.Tm per 20μm section. (D) S.Tm CFU counts in cecal mucosa tissue. In A, C, and D each data point corresponds to one animal. Line at median. (C-D) Mann-Whitney U-test (**p<0.01). (E) Representative micrographs of the extensively washed cecal mucosa. Blow-up in rightmost panel highlights S.Tm^ΔsipA on the epithelial surface (white arrow heads). Lu.—Lumen; Ep.—Epithelium. Scale bar: 50μm. (F) Barcoded consortium infections reveal the differential dependence on bacterial virulence factors for S.Tm invasion of epithelial cell lines vs. the gut absorptive epithelium in mice. Relative abundance of the indicated barcoded S.Tm strains in the intracellular population in HeLa cells (left panel; 20min infection at MOI 0.2–2) and in vivo in the cecal mucosa of Nlrc4^-/- mice (right panel; infected as in A above). Bars correspond to mean +/- SD of six replicate infections pooled from two different occasions (left panel) or infection of seven replicate mice (right panel). Each replicate is indicated by a circle symbols. One-way ANOVA with Dunnett´s test (n.s., not significant; *p<0.05, ***p<0.001).

https://doi.org/10.1371/journal.ppat.1008503.g002

We first infected Nlrc4^-/- mice for 18h with S.Tm^wt or S.Tm^ΔsipA harboring the pssaG-GFP reporter. The strains colonized the gut lumen equally (Fig 2A), but S.Tm^ΔsipA again exhibited ~100-fold lower total numbers of epithelium-lodged GFP+ bacteria (Fig 2B and 2C). Enumerating the number of infected cells per section instead of the total S.Tm load yielded similar results, i.e. 100-200-fold lower numbers of epithelial cells infected by S.Tm^ΔsipA (S3A Fig). Moreover, virtually identical results were also obtained upon gentamycin treatment of the mucosal tissue followed by plating of total intracellular bacteria (Fig 2D). Finally, staining of permeabilized cecal sections with anti-Salmonella-LPS antibodies revealed plenty of tissue-residing S.Tm^wt (Fig 2E), whereas S.Tm^ΔsipA invasion foci were virtually absent from the epithelial tissue (Fig 2E). Instead, S.Tm^ΔsipA were found enriched on the epithelial surface (Fig 2E, rightmost panel and insert). These results exclude that the effect of SipA on epithelial S.Tm loads in vivo can be explained by altered reporter maturation, or by a SipA effect on replication (although we do not refute that such effects could also exist). Of further note, intraepithelial S.Tm foci on average contain only a low number (mean ~2) of bacteria during the first 12-18h of infection ([30]; see also Fig 5E below). This means that the frequency of invasion events, rather than intraepithelial replication, predominantly governs the intraepithelial S.Tm load during early gut infection. Taken together, our results support that SipA deletion results in normal S.Tm gut lumen colonization, normal approach of and binding to the gut epithelium, but a profound epithelial cell invasion defect.

In vivo infections are subject to large animal-to-animal variations. Furthermore, in infections with one individual mutant per mouse, mutants attenuated at an early step of the infection process may face delayed onset of host defense and thereby grow or survive differently than S.Tm^wt. This could complicate scoring of attenuation phenotypes in vivo. To substantiate the contribution of S.Tm effectors to epithelial cell invasion under internally controlled conditions, we employed our recently developed method for barcoded consortium infections [19]. Unique inert 40-nucleotide tags (informed by [58]) were placed on the bacterial chromosome of each strain of interest and infections conducted with a mixed inoculum comprising equal amounts of each strain. Quantitative PCR of genomic DNA was employed to quantify the relative abundance of each tag in enrichment cultures of the input (inoculum or luminal content) and output (intracellular) bacterial populations (see materials and methods for details).

For the consortium infections, we employed seven isogenic barcoded strains: S.Tm^wt-tag A, S.Tm^ΔsipA-tag B, S.Tm^ΔsopBEE2-tag C, S.Tm^Δ4-tag D, S.Tm^ΔinvG-tag E, and the additional control strains S.Tm^Δspi-4-tag F, and S.Tm^{ΔinvGΔspi-4}-tag G (S1 Table). These strains were mixed in a 1:1:1:1:1:1:1 ratio and used as inoculum for infections in both cultured epithelial cell lines and the in vivo model. Quantitative PCR showed similar abundances of each strain in the inoculum (S3B Fig). However, in the population successfully invading HeLa cells (MOI 0.2–2; 20min infection), the relative strain abundance was shifted significantly. S.Tm^ΔsipA-tag B and S.Tm^Δspi-4-tag F were present at roughly equal levels than S.Tm^wt-tag A (Fig 2F, left panel). By contrast, S.Tm^ΔsopBEE2-tag C, S.Tm^Δ4-tag D, S.Tm^ΔinvG-tag E, and S.Tm^{ΔinvGΔspi-4}-tag G were all close to undetectable (<1% of total bacterial population). Similar results were also obtained in infections of m-CIc12 cells on plastic and of polarized Caco-2 C2Bbe1 cells grown atop Transwell inserts (S3C and S3D Fig). Using a less complex consortium, we at shorter infection times (7-10min) detected ~1.5-2-fold attenuated invasion of S.Tm^ΔsipA in polarized Caco-2 C2Bbe1 (S3E Fig). These results are in agreement with results from automated microscopy and bacterial plating assays above (Fig 1A, S1A Fig, S1D Fig and S2F and S2G Fig).

Next, we infected Nlrc4^-/- mice with the same seven-strain consortium and extracted the luminal and epithelial tissue-residing bacterial populations. This yielded the expected total bacterial loads in both compartments (~10⁹ S.Tm/g cecum content, S3F Fig; and ~10⁶ S.Tm in tissue/cecum, S3G Fig). S.Tm^wt-tag A outperformed the other strains with respect to epithelial invasion (~55% of the intracellular population; Fig 2F right panel). S.Tm^Δspi-4-tag F exhibited a modest attenuation (~23% of intracellular population) compared to the wild-type. Importantly, deletion of SipA (S.Tm^ΔsipA—tag B) again resulted in a dramatic loss in invasiveness (≤1% of total intracellular population), whereas the strain lacking the ruffle-inducers (S.Tm^ΔsopBEE2—tag C) performed markedly better (~16% of total intracellular population) (Fig 2F right panel). Taken together, multiple experimental approaches demonstrate that S.Tm invasion of the murine gut absorptive epithelium critically depends on TTSS-1 translocation of the primordial effector SipA. This contrasts starkly to observations of the invasion process in cultured epithelial cell lines.

S.Tm invades gut absorptive epithelial cells through discreet entry structures

The results above (Figs 1 and 2) indicate that SipA is a key driver of epithelial cell invasion in vivo, in the absence or presence of SopBEE2. Importantly, previous work has suggested that SipA on its own is incapable of inducing large ruffles in cultured cell lines (e.g. [26,59]). The strong SipA-dependence for gut epithelium entry raises the question whether the model for S.Tm invasion through large SopBEE2-dependent ruffles applies to the in vivo scenario. We hypothesized that the primary, differentiated, polarized, and neighbor-connected nature of absorptive epithelial cells in vivo steer S.Tm invasion towards a SipA-dependent, and away from a ruffle-dependent, entry mechanism. We therefore examined the presence and morphology of entry structures around invading S.Tm in cell lines exhibiting different degrees of polarization, and in the mouse gut.

We began by characterizing S.Tm entry structures in MDCK cells, cultured in parallel either as flat-growing or polarized cell layers on plastic. The cells were infected with S.Tm^wt expressing constitutive GFP (pM965; S1 Table) and infections terminated by fixation. We consistently noted induction of ~3.5–8μm high actin ruffles in non-polarized MDCK cells (Fig 3A and 3B). Polarized MDCK cells produced significantly smaller entry structures (~2–4.5 μm height) in response to S.Tm^wt/pGFP (Fig 3A and 3B). In agreement with previous work [27], these ruffles were also shaped differently, often with a circular appearance when viewed from the top. Similar results were obtained with an S.Tm strain expressing a SopE-M45 reporter protein (S1 Table), which allowed focusing the analysis on host cells that recently experienced delivery of TTSS-1 effectors (Fig 3B). Moreover, when m-ICc12 and Caco-2 C2Bbe1 cells were grown as semi-polarized/polarized monolayers atop Transwell inserts (see growth conditions in S2D and S2E Fig), they produced smaller ruffles upon S.Tm infection, as compared to their subconfluent non-polarized counterparts (Fig 3C–3E). These data show that polarization of epithelial cells reduces their propensity to generate large ruffles in response to S.Tm docking and effector translocation.

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Fig 3. S.Tm-induced entry structures differ between epithelial cell lines and the absorptive gut epithelium in mice.

(A-E) Epithelial cell polarization status affects the size of S.Tm-induced actin ruffles. (A) Confocal microscopy Z-stack visualizations of non-polarized and polarized MDCK cells infected with S.Tm^wt/pGFP. Scale bar: 10μm. (B) Quantification of cortical actin height at invasion foci in MDCK cells infected with S.Tm^wt/pGFP or S.Tm^wt/psopE-M45. (C) Representative maximum intensity projection micrographs of non-polarized and semi-polarized m-ICc12 cells grown atop Transwell inserts and infected with S.Tm^wt/pmCherry (green pseudocoloring). White dashed line indicate S.Tm-induced entry structures. Scale bars: 10μm. (D-E) Quantification of S.Tm-induced ruffle area in (D) non-polarized and semi-polarized m-ICc12 cells and (E) non-polarized and polarized Caco-2 C2Bbe1 cells grown atop Transwell inserts. (F-I) Intact actin brush border without signs of actin ruffle formation in the S.Tm-infected gut absorptive epithelium in vivo. (F-G) Wild-type mice were orally infected with S.Tm^wt /pssaG-GFP for 6h. (F) Representative micrographs of the cecal epithelium. Blow-ups show magnifications of boxed regions. Lu.—Lumen; Ep.—Epithelium. White arrows indicate the apical actin brush border of an infected cell. Scale bar: 10μm. (G) Quantification of actin brush border height in infected cells and uninfected neighbors. (H-I) Rag1^-/- mice (used since detection of M45 relies on a mouse monoclonal) were orally infected with S.Tm^wt/psopE-M45 for 8h. (H) Representative micrographs of a SopE-M45 positive focus in the cecal epithelium. Blow-up shows magnification of boxed region. Lu.—Lumen. White arrow indicates M45-positive bacterial focus. Scale bar: 10μm. (I) Quantification of actin brush border height in infected cells and uninfected neighbors. Each data point in B, D, E, G, and I corresponds to one (non-infected or infected) cell. Bar or line represents mean. Results are representative for two to three experiments in each case. Mann-Whitney U-test (n.s., not significant; **p<0.01; ***p<0.001). (J-L) Different ultrastructural appearance of S.Tm entry structures in epithelial cell lines and the gut absorptive epithelium in mice. (J-K) Representative SEM micrographs of (J) HeLa cells and (K) m-ICc12 cells infected with S.Tm^wt for 6-10min at MOI 400. (L) Representative SEM micrographs of the cecal epithelium of mice infected with S.Tm^wt. Arrows indicate S.Tm. Scale bars as indicated in each panel.

https://doi.org/10.1371/journal.ppat.1008503.g003

High loads of luminal bacteria complicate the use of constitutive reporters to visualize epithelial cell invasion in vivo. Using S.Tm/pssaG-GFP in mice, we did not observe overt perturbation of the apical actin brush border of infected epithelial cells at the resolution of light-microscopy (Fig 3F and 3G, S4A Fig). This could imply that i) no ruffles are forming, or that ii) in vivo entry structures are exquisitely short-lived and the apical actin returns to normal before the ssaG-GFP-reporter produces visible fluorescence. To resolve this uncertainty, we again utilized the S.Tm^wt/psopE-M45 reporter strain. Of note, translocated effector proteins can by this approach be detected within less than a minute of TTSS-1 secretion [60]. Indeed, SopE-M45 could be detected as a deposit in occasional infected epithelial cells in vivo (Fig 3H and S4B Fig). Since this effector exhibits a short half-life within host cells (half-life << 1h; [61]), the approach allowed us to focus the analysis of entry structures to epithelial cells that had recently experienced TTSS-1 effector translocation. Notably, we again did not detect any pronounced perturbations of the actin brush border of infected cells compared to uninfected neighbors (Fig 3H and 3I, S4B Fig).

Next, we used scanning electron microscopy (SEM) to investigate the ultrastructural appearance of S.Tm entry sites. As expected, S.Tm inoculums contained rod-shaped ~0.5–1μm wide and ~2μm long bacteria with peritrichous flagella (S5A Fig). In HeLa cells, large membrane ruffles accompanied 48% of all cell-associated S.Tm^wt (N = 128) (Fig 3J panel ii-iv, S5B Fig). The remainder of the events did not involve direct contact between the bacterial body and the host cell, and thus likely represent pre-invasion stages (Fig 3J, panel i). S.Tm-induced ruffles were typically ~10–20μm in diameter and featured large lamellipodial structures (Fig 3J, panels ii-iii, S5B Fig), combined with filopodial protrusions (Fig 3J, panel iv; S5B Fig). Infected mouse m-ICc12 cells produced somewhat smaller ruffles, but still containing both lamellipodial and filopodial elements and reaching diameters of up to ~10μm (Fig 3K).

A parallel SEM analysis of the intact murine gut epithelium (of S.Tm^wt-infected mice) provided no evidence of expansive membrane ruffles. Instead, S.Tm^wt-associated gut epithelial cells featured either no signs of cell surface perturbation (Fig 3L, panel i; S5C Fig, top right panels), or only discreet ultrastructural effects (Fig 3L, panels ii-iv; S5C Fig, bottom panels). Where host cell binding was evident, S.Tm engaged in multivalent interactions with, and distortion of, proximally located microvilli (Fig 3L, panel ii; S5C Fig, bottom panels). In some further progressed invasion events, a rim of elongated, bent, and deformed microvilli emerged around the bacterium (Fig 3L, panel iii; S5C Fig, bottom panels). Finally, S.Tm captured in late stages of invasion displayed a “sinking-in” appearance surrounded by a mix of normal and distorted microvilli (Fig 3L, panel iv; S5C Fig, bottom panels). Importantly, no large lamellipodial ruffles were observed (scrutiny of the cecal mucosa in N = 5 mice). From these data, we conclude that S.Tm enters the intact gut absorptive epithelium through discreet entry structures, distinct from the expansive SopBEE2-dependent ruffles observed in epithelial cell lines. This agrees with an entry mechanism dominated by TTSS-1 delivery of the primordial effector SipA (Figs 1 and 2). We term this mode of S.Tm entry into the murine gut epithelium “discreet-invasion”.

SipA drives ruffle-independent invasion into a tight vacuolar compartment

We next aimed to resolve the dynamic features of SipA-driven epithelial cell invasion at high temporal resolution. We began by infecting non-polarized epithelial cell lines, i.e. sub-confluent MDCK and HeLa cells expressing LifeAct (for actin visualization), with fluorescent S.Tm strains. At the end of the time-series, fixation and anti-S.Tm-LPS staining without prior permeabilization allowed us to determine which pathogen-host cell encounters had led to successful invasion.

Initial experiments showed that S.Tm^wt always entered non-polarized MDCK cells (N = 157/157) and HeLa cells (N = 263/263) through clearly visible actin ruffles ((Fig 4A, 4B and 4E and S1 Movie (non-polarized MDCK) and S2 Movie (HeLa)). Ruffle duration (from 4min to persistence over the whole 24min series) and size (~5–15μm diameter in MDCK cells) varied greatly, however. The strain invading through SipA (i.e. S.Tm^ΔsopBEE2) has a poor capacity to enter cultured cell lines (Fig 1A–1C), which prompted us to carry out the corresponding live experiments at a high MOI over a longer time period (MOI 500, 40min). Nevertheless, all S.Tm^ΔsopBEE2 invasion events detected in both cell types occurred in the complete absence of actin ruffles (Fig 4C–4E and S3 Movie).

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Fig 4. SipA drives S.Tm epithelial cell invasion without triggering higher order actin meshworks.

(A-E) By contrast to S.Tm^wt, S.Tm^ΔsopBEE2 invades non-polarized epithelial cells in the absence of visible ruffles. (A-B) Non-polarized LifeAct-expressing MDCK cells (red) were infected with S.Tm^wt/pmCherry (green) at MOI 50. (A) Representative micrograph of cells fixed at the end point (24min p.i.). Successful invasion was scored by α-S.Tm LPS-staining prior to permeabilization. R–ruffle ;? —ambiguous entry event revealed as a ruffle in the live series. (B) Live imaging series preceding A. Arrow/Arrow heads labelled R indicate extensive and transient ruffles, respectively. (C-D) MDCK cells as in A-B were infected with S.Tm^ΔsopBEE2/pmCherry (green) at MOI 500. (C) Representative micrograph of cells fixed and stained at the end point (40min p.i.). (D) Live imaging series preceding C. Arrow head indicates a ruffle-less entry focus. Scale bars in A-D: 20μm. (E) Live imaging-based quantification of the presence/absence of ruffles at S.Tm^wt and S.Tm^ΔsopBEE2 entry sites in non-polarized MDCK (left; N_tot = 164 invasion events) and HeLa (right graph; N_tot = 271 invasion events analyzed). (F-H) Ultrastructural underpinnings of SipA-driven epithelial cell invasion. Cryo-electron tomograms of HeLa cells infected with S.Tm^wt (F-G; N_{tot tomograms} = 20, frozen at 14min p.i.), or S.Tm^ΔsopBEE2 (H; N_{tot tomograms} = 13; frozen at 40min p.i.). Shown in each case is a 15-17nm slice of a tomogram and the respective segmentation. R—ruffle; OM—outer membrane; IM—inner membrane; bCP—bacterial cytoplasm; hCP—host cell cytoplasm; hCM—host cell membrane; A—actin filaments; white pseudo-coloring—host cell membrane; red pseudo-coloring—bacterial membrane. Scale bars in F-H: 100 nm.

https://doi.org/10.1371/journal.ppat.1008503.g004

To survey the impact of epithelial cell polarization, we visualized a large number of S.Tm^wt invasion events (N = 524) in real-time in polarized LifeAct-expressing MDCK cells. Microscopy of samples fixed at the end point of infection highlighted abundant intracellular S.Tm (S6A Fig). Analysis of the preceding live-imaging series revealed that a majority of invasion events (94,8%) were surrounded by the pronounced actin signal enrichment expected from a ruffling response (S6A–S6C Fig; indicated by arrows; S4 Movie). Notably, however, a fraction (5,2%) of invasion events displayed no signs of actin ruffles around the invading bacterium at any time of the movie (S6A–S6C Fig; encircled in white; S4 Movie). These data indicate that wild-type S.Tm can in fact enter polarized epithelial cells without triggering marked actin-dependent ruffles. Analogous experiments showed that the S.Tm^ΔsopBEE2 strain always entered polarized MDCK cells without the emergence of actin ruffles (N = 24/24) (S6D–S6F Fig; indicated by arrow heads; S5 Movie). Hence, SipA drives ruffle-independent epithelial cell invasion regardless of epithelial host cell polarity or context (Fig 4C–4E, S6D–S6F Fig).

Cryo-electron tomography of the thin edges of plunge-frozen HeLa cells was used to determine the ultrastructural underpinnings of the wild-type and “SipA-only”-driven invasion processes in a near-native state. The cryo-tomograms illustrated abundant actin bundles underneath S.Tm^wt captured at early stages of host cell binding (S6 Movie and Fig 4F). For events captured at a later stage of invasion, these bundles were replaced by a multidirectional meshwork of actin filament-rich protrusions around the bacterium (S7 Movie and Fig 4G; red pseudo-coloring delineates the bacterial membrane and white pseudo-coloring the host cell membrane). Again by sharp contrast, invading S.Tm^ΔsopBEE2 bacteria were found within a tightly wrapped and smooth membrane compartment (S8 Movie and Fig 4H). Hence, the S.Tm^ΔsopBEE2 strain, which invades specifically through SipA, enters non-polarized epithelial host cells by sinking into a tight vacuole without inducing complex higher-order actin meshworks. This is especially notable since non-polarized epithelial cells are highly permissive for ruffling if exposed to bacteria expressing SopB, SopE and/or SopE2. Taken together, our time-lapse and 3D-reconstruction data provide a direct link between the primordial TTSS-1 effector SipA and a discreet-invasion mode for epithelial cell entry.

Non-cooperative S.Tm invasion into the murine gut absorptive epithelium

Cooperative invasion, i.e. that an actively invading bacterium promotes the entry also of bystander bacteria, is prevalent in cultured cell lines infected with S.Tm [5,18,19]. Expansive membrane ruffles elicited by the primary invader generates physical obstacles where secondary motile bacteria get entangled and taken up [5]. Cooperative invasion depends on the likelihood of secondary bacteria finding a ruffle and will therefore increase in frequency with higher MOI, larger ruffle size, and/or longer ruffle duration [5,18,19]. As such, cooperative invasion provides a functional readout for S.Tm-elicited host cell ruffling responses.

In the mouse cecum, the mucus barrier covers the crypts, whereas the top ~50% of the epithelial layer is in contact with motile luminal S.Tm [62]. Based on this prior knowledge, we used confocal microscopy to estimate the effective MOI in the cecum at 12h p.i. (in Nlrc4^-/- mice to prevent epithelium distortion). We quantified the total luminal S.Tm population and the number of accessible epithelial cells per section. S.Tm were evenly spaced over the gut lumen cross-section with a modest enrichment at the epithelial border (Fig 5A). As expected, only few bacteria localized within crypts, while S.Tm were frequently found in contact with the differentiated part of the gut epithelium (Fig 5B). Repeated analysis resulted in an MOI estimation of 91+/-20 (mean+/-SD; Fig 5C). It should here be noted that luminal S.Tm loads in wild-type and Nlrc4-deleted mice are equal during early infection (Figs 1B and 2A) [30]. Moreover, the gut luminal pathogen population reaches a stable plateau of colonization (~10⁸−10⁹ CFUs/gram content) already at 6-8h p.i. (S1J Fig) [30]. Hence, the naïve mouse cecal epithelium experiences close contact with a dense and motile luminal S.Tm population for several hours during acute infection.

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Fig 5. Cooperative S.Tm invasion occurs frequently in epithelial cell lines, but not in the absorptive gut epithelium of mice.

(A-C) S.Tm Multiplicity of Infection (MOI) in the gut of (Sm-pretreated) orally infected mice. Inflammasome-deficient (Nlrc4^-/-) mice were orally infected with S.Tm^wt for 12h. (A) Representative stitched image of an entire cross-section of the infected murine cecum. Scale bar: 500μm. (B) Representative up-close micrograph of the infected cecum lumen and mucosa. Lu.—Lumen; Ep.—Epithelium; Cr.—Crypt. Scale bar: 20μm. (C) Microscopy-based quantification of the MOI in the infected mouse cecum. Bar correspond to mean +/- SD of replicate infections in four mice. (D-F) S.Tm co-invasion occurs rarely in the mouse gut. Inflammasome-deficient (Nlrc4^-/-) mice were orally infected with a 1:1 mix of S.Tm^wt/pssaG-mCherry and S.Tm^wt/pssaG-GFP strains for 12h. (D) Representative micrographs of the cecal epithelium. Blow-up shows magnification of boxed region. Lu.—Lumen. White arrows indicate one-coloured invasion foci; arrow head labelled “co-inv” indicates a mixed invasion focus. Scale bar: 10μm. (E) Quantification of the distribution of bacterial numbers within S.Tm invasion foci. (F) Quantification of the frequency of single-colour and mixed colour invasion foci in vivo. Bars in E-F correspond to mean +/- SD of replicate infections in four mice (N_tot = 1055 invasion events analyzed). (G-J) Prevalent cooperative S.Tm invasion in epithelial cell lines, but not in the absorptive gut epithelium in vivo. (G-H) Infection of (G) HeLa and (H) m-ICc12 cells with a 1:1 mix of S.Tm^wt/pssaG-mCherry and S.Tm^ΔinvG/pssaG-GFP strains for 1h at MOI 80. Representative micrographs. Blow-ups show magnifications of boxed regions. Arrow heads labelled “coop” indicate cooperative invasion foci. Scale bar: 20μm. (I) Inflammasome-deficient (Nlrc4^-/-) mice were orally infected with a 1:1 mix of S.Tm^wt/pssaG-mCherry and S.Tm^ΔinvG/pssaG-GFP strains for 12h. Representative micrographs of the cecal epithelium. Blow-up shows magnification of boxed region. Arrow points to a rare S.Tm^ΔinvG/pssaG-GFP invasion focus. Scale bar: 20μm. (J) Quantification of the frequency of cooperative invasion foci in HeLa, m-ICc12 cells (infection parameters as in G-H) and the absorptive gut epithelium in vivo (infection parameters as in I). Data points correspond to mean +/- SD of three replicate infections in HeLa and m-ICc12 cells, and to mean +/- SD of five mice (N_tot = 578 in vivo invasion events analyzed), respectively.

https://doi.org/10.1371/journal.ppat.1008503.g005

We infected Nlrc4^-/- mice with a 1:1 mix of two differentially labelled wild-type S.Tm strains (S.Tm^wt/pssaG-mCherry and S.Tm^wt/pssaG-GFP) to begin assessing cooperative invasion frequency. At 12h p.i. ~55% of all epithelial invasion foci carried only one bacterium, ~30% carried two bacteria, and the remaining foci carried three bacteria or more (Fig 5D and 5E). Most notably, only ~5% of all invasion foci carried a mix of green and red bacteria (Fig 5D–5F; N_tot = 1055 foci in 4 mice analyzed). This subfraction of invasion foci could either have resulted from co-invasion (i.e. two active invasion events into the same host cell, occurring in parallel or in sequence), or alternatively from true cooperative (helped) invasion. In either case, the results show that at an estimated MOI of 91+/-20 in the mouse gut, cooperative epithelial cell invasion is at best rare.

To estimate the frequency of true cooperative invasion events, we adapted the dual-colored mixed inoculum to include one actively invading strain (S.Tm^wt/pssaG-mCherry) and one strain incapable of TTSS-1-mediated active entry (S.Tm^ΔinvG/pssaG-GFP; >1000-fold reduced invasion capacity in single strain infections; S1L Fig). A mixed invasion focus could with this setup only arise if the S.Tm^wt strain promoted cooperative entry of S.Tm^ΔinvG. As reference, the mixed inoculum was used to infect cultured HeLa and m-ICc12 cells at MOIs spanning across the range noted in vivo (MOI 20–320, 1h infection). HeLa cells produce expansive S.Tm-induced ruffles (Fig 3J), and as expected cooperative invasion was highly prevalent at all MOIs tested (Fig 5G–5J). At an MOI close to the in vivo estimate (MOI 80) >80% of all invaded cells carried S.Tm of both colors (Fig 5J). Mouse m-ICc12 cells produce somewhat smaller ruffles (Fig 3K), and consequently cooperative invasion was less common than in HeLa cells, but still comprised ~40% of all invasion events at MOI 80 (Fig 5H–5J). By stark contrast, we did not observe a single case of cooperative epithelial cell invasion in the mouse cecum (Fig 5I and 5J; N_tot = 578 foci in 5 mice analyzed). This means that even when the gut luminal S.Tm population supports a high MOI in close contact with the mucosa for several hours (Fig 5A–5C), cooperative S.Tm entry does not occur (Fig 5J). These data provide functional support for S.Tm discreet-invasion into the murine gut absorptive epithelium.

S.Tm discreet-invasion preferentially occurs proximal to cellular junctions

While studying the impact of apical S.Tm^wt binding to the gut epithelium in vivo, we noted that bacteria frequently localized to the cell–cell junctional zones, separating individual epithelial cells (e.g. Fig 3H and 3L panel i, S4B Fig). These circumstantial observations prompted us to investigate if S.Tm discreet-invasion exhibits preference for specific apical locations. To study the surface-binding S.Tm population in vivo in isolation, luminal bacteria were removed by repeated gentle washing of infected tissue, prior to fixation and staining of the remaining adherent S.Tm^wt (Fig 6A). Again, we noted only modest perturbations of the local actin brush border proximal to attached S.Tm. Moreover, S.Tm surface binding exhibited a highly non-random pattern; ~80% of all bacteria could be found within a 2μm distance from the closest cell–cell junction (Fig 6B and 6C).

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Fig 6. Cellular and subcellular S.Tm targeting preferences in the gut epithelium.

(A-F) S.Tm preferentially binds the apicolateral region during first interactions with gut absorptive epithelial cells. (A-C) Wild-type mice were orally infected with S.Tm^wt for 9h. (A) Representative micrograph of the extensively washed cecal mucosa. Arrow head indicates a bound S.Tm. Scale bar: 10μm. (B) Experimentally determined dimensions of cecal absorptive epithelial cells (mean+/-SD). (C) Quantification of the distance between epithelial surface-bound S.Tm and the nearest cell–cell junction. Data in B-C correspond to mean+/-SD of replicate infections in four mice. (D-E) Representative SEM micrographs of the cecal epithelium of Nlrc4^-/- mice infected with S.Tm^wt for 12h. In D arrow heads indicate cell–cell junctions; arrow points to a bound S.Tm. In E, blue shading in the blow-up indicate zones used for quantification. Scale bars in D-E: 2μm. (F) Quantification of the distribution of epithelial surface-bound S.Tm^Δ4 in the cecal epithelium of Nlrc4^-/- mice infected for 18h. Bars correspond to mean +/- range of replicate infections in two mice. (G-I) S.Tm preferentially invades polarized cultured epithelial cells at apicolateral sites. Polarized LifeAct-expressing MDCK cells (red) were infected with S.Tm^wt/pmCherry (green) at MOI 50. (G) Representative maximum intensity projection micrograph of cells fixed at the end point (14min p.i.). (H) The first frame in the live imaging series where the indicated S.Tm (white arrow) could be found. Blue shading in the blow-up indicate zones used for quantification. Scale bars in G-H: 10μm. (I) Quantification of the distribution of epithelial cell invasion events in G-H. Bars correspond to mean +/- SD of four replicate MDCK infections. (J-L) Preferential S.Tm invasion of goblet-cell neighboring absorptive epithelial cells. (J) Representative SEM micrograph of the cecal epithelium of a wild-type mouse infected with S.Tm^wt for 8h. Arrow heads indicate extruding mucus from a goblet cell. arrow points to a bound S.Tm. Scale bar: 2μm. (K-L) Wild-type mice were orally infected with S.Tm^wt/pssaG-GFP for 6h. (K) Representative micrographs from confocal Z-stacks. Lu.—Lumen; Ep.—Epithelium; G—Goblet cell. White arrows point to S.Tm invasion foci. Scale bar: 10μm. (L) Quantification of the frequency of S.Tm invasion into goblet cell neighboring and non-neighboring epithelial cells. Bars correspond to mean +/- SD of replicate determinations in ten cecal tissue sections. Mann-Whitney U-test (**p<0.01).

https://doi.org/10.1371/journal.ppat.1008503.g006

We adapted the procedure for SEM imaging of the gut epithelial surface from the luminal side (Fig 6D). To quantify the distribution of bound S.Tm, the surface of each epithelial cell was subdivided into three zones of equal area. Zone 1 covered the junction-proximal part, zone 2 the intermediate part, and zone 3 the mid part of the cell surface (Fig 6E). The center of each bound S.Tm was subsequently mapped onto these zones. To increase the number of bacteria that could be observed in this transient pre-invasion state, we infected mice with S.Tm^Δ4, which lacks all the major TTSS-1 effectors, but remains competent for host cell binding. A marked enrichment of S.Tm binding was noted in zone 1, which carried a higher fraction of all bound bacteria than zone 2+3 combined (Fig 6F).

To test if not only binding, but also S.Tm invasion, exhibited preference for the apicolateral zone in polarized epithelial cell layers, we next performed a similar analysis in live polarized MDCK cells expressing LifeAct. Fixation and staining for S.Tm-LPS without permeabilization was used at the end point of the infection to focus the analysis on successful invasion events (Fig 6G). Each event was traced back to the moment of entry in the live series, and the apical host cell membrane subdivided into zones as above. In full agreement with results from the binding experiments in vivo, a majority of S.Tm invasion events mapped to zone 1 (Fig 6H and 6I). Based on these data, we conclude that an apicolateral surface region represents a hotspot for S.Tm binding and invasion of the polarized gut absorptive epithelium.

By contrast to homogeneous epithelial cell lines, the intact gut epithelium comprises multiple cell types in addition to absorptive epithelial cells. Of these, mucus-producing goblet cells make up ~12% of the total cell-count in the murine cecum. In the SEM analysis, we frequently observed that epithelium-adherent S.Tm localized close to the junctional zones between an absorptive epithelial cell and its goblet cell neighbor (Fig 6J). The S.Tm/pssaG-GFP reporter strain was therefore again used to examine the neighborhoods of epithelial cells targeted by S.Tm invasion. The results revealed a ~5-fold enrichment of S.Tm invasion events into goblet-cell-neighboring epithelial cells, as compared to goblet cell non-neighbors (Fig 6K and 6L). This points to a non-random targeting of S.Tm epithelial cell discreet-invasion both at the cellular (i.e. goblet-cell neighbors) and subcellular (junctional zone-proximal) level.

Ethics statement

All animal experiments were performed in accordance to the Swiss Federal Government guidelines in the animal experimentation law (SR 455.163 TVV). The protocols used were approved by the Cantonal Veterinary Office of the canton Zürich, Switzerland (Kantonales Veterinäramt ZH licenses 223/2010, 222/2013, and 193/2016).

Bacterial strains, plasmids and culture conditions

All S.Tm strains used in this study were isogenic derivatives of SL1344 [76]. Strains and bacterial plasmids are detailed in S1 Table. For infections, S.Tm strains were grown in LB broth/0.3M NaCl, supplemented with 50μg/mL streptomycin (AppliChem), 50μg/mL ampicillin (AppliChem), or 12.5μg/mL chloramphenicol (Sigma Aldrich) for 12h at 37°C in a rotating wheel incubator. Cultures were diluted 1:20 in the same broth w/o antibiotics and cultured for an additional 4h at 37°C. For epithelial cell line infections, the inoculum was diluted in tissue culture medium to achieve the indicated MOI. For mouse infections, the inoculum was washed in sterile phosphate-buffered saline pH 7.4 (PBS; Gibco or Amimed) and reconstituted in PBS to a concentration of ~10⁹ CFUs/ml.

Epithelial cell line culture and infections

HeLa (CCL-2; ATCC), MDCK (subclone Pf´ [37]; kind gift from Michael Hensel), and MDCK-LifeAct-GFP transfectants cells were maintained in DMEM (Gibco) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Invitrogen). m-ICc12 cells [51] were cultured in DMEM/F12 (Invitrogen) supplemented with 5μg/ml insulin (Invitrogen), 50nM dexamethasone (Sigma Aldrich), 60nM sodium selenite (Sigma Aldrich), 5μg/ml bovine apo-transferrin (Sigma Aldrich), 1nM triiodothyronine (Sigma Aldrich), 60ng/ml EGF (Sigma Aldrich), 2mM glutamine (Invitrogen), 12.5mM D-glucose (Sigma Aldrich), 20mM hepes (Gibco), and 2% heat-inactivated FCS. Caco-2 C2Bbe1 (CRL-2102; ATCC) cells were maintained in DMEM supplemented with 10μg/ml human Transferrin (Sigma Aldrich), and 10% heat-inactivated FCS. For growth atop Transwell inserts, Caco-2 C2Bbe1 cells were seeded out in DMEM supplemented with 1:1000 Mito+ serum extender (Corning), and the medium exchanged at 24h post-seeding for Enterocyte Differentiation Medium (Corning) with Mito+ serum extender. m-ICc12 cells were grown atop Transwell inserts in their regular culture medium. Transwells were pre-coated with Collagen-1 (~30μg/cm2; rat-tail Collagen-1; Corning) prior to cell seeding. The status of monolayers atop Transwell inserts was followed by TEER measurement (EVOM² voltohmmeter with STX2 chopsticks electrode; World Precision Instruments). For live imaging, HeLa cells were transiently transfected with LifeAct-mCherry (mCherry-Lifeact-7; kind gift from Michael Davidson; addgene construct #54491) using Lipofectamine^TM 2000 (Thermo Fisher) according to the manufacturer’s protocol. All culture media used was supplemented with 50μg/mL streptomycin sulphate or with PenStrep (Gibco), but antibiotics were omitted in preparation for infection. Cell lines were kept at 37°C in a CO₂-buffered incubator and infected by adding a given S.Tm strain for the indicated time. For enumeration of S.Tm invasion efficiency by plating, infected cells were washed with fresh culture medium, incubated with culture medium containing 200μg/ml gentamicin (AppliChem) ≥40min, washed with PBS, lysed in 0.1% sodium deoxycholate (Sigma Aldrich), and serial dilutions of the lysates plated on LB agar with appropriate antibiotics.

Mice and in vivo infections

Mice were bred and kept in individually ventilated cages under specific pathogen free conditions (RCHCI and EPIC facilities, ETH Zürich). Wild-type C57BL/6 mice were originally from Charles River, Rag1^-/- (B6.129S7-Rag1tm1Mom/J) from Jackson Laboratory, and Nlrc4^-/- mice have been described elsewhere [57]. S.Tm infections were performed as detailed in [33]. In brief, ~8–12 week old mice were treated with 25mg streptomycin sulphate per oral gavage. This step is required to suppress gut microbiota colonization resistance and thereby permit luminal expansion of S.Tm. 24h later, mice were infected per oral gavage with 5x10⁷ CFUs of the indicated S.Tm strain. Bacterial loads in gut lumen content and organs were monitored by plating homogenized samples on MacConkey agar (Oxoid) with 50μg/ml streptomycin. Plating of cecum tissue was done subsequent to a 30min incubation in PBS/400μg/ml gentamicin, six rigorous washes in excess PBS, and tissue homogenization. For histopathology scoring, cecum tissue was frozen in optimum cutting temperature medium (OCT; Tissue-Tek). 5μm cross-sections were air dried and stained with hematoxylin and eosin. Histopathology scoring was done blindly as described [33]. Briefly, a score was assigned based on the degree of submucosal edema, polymorphonuclear leukocyte infiltration, goblet cell numbers, and epithelial damage. The possible scores range from 0 (uninflamed) to 13 (maximum inflammation).

Fluorescence microscopy of fixed cell lines and tissues

Uninfected or S.Tm-infected epithelial cell lines grown as indicated were fixed in 2–4% room-temperatured paraformaldehyde (PFA)/4% sucrose or in ice-cold methanol (for tubulin staining). Cells were permeabilized with PBS/0.1% TritonX-100 (Sigma Aldrich) or PBS/0.1% saponin (Calbiochem), and unspecific binding blocked in PBS/10%FCS. After staining with the indicated antibodies (detailed below), cells were mounted with Mowiol (Calbiochem) and imaged the following day. Mouse cecum tissue (or other intestinal segment if indicated) was fixed in 4% PFA, saturated in PBS/20% sucrose, embedded in OCT, flash-frozen, and stored at −80°C. In cases where luminal bacteria were removed, the intestinal segment was opened longitudinally and washed extensively in PBS before fixation and embedding. 10–20μm cryosections were placed on glass slides, air dried, rehydrated in PBS, permeabilized with PBS/0.5% Triton X-100, and blocked in PBS/10% normal goat serum (NGS; Vector Labs). After immunostaining, the samples were mounted with Mowiol and imaged the following day. Antibodies and staining reagents used in this study were α-EpCAM/CD326 (G8.8, Biolegend), α-gp135 (3F2/D8-s; DSHB), α-ICAM-1/CD54 (clone 3E2, Becton Dickinson), α-M45 (purified from hybridoma), α-S.Tm LPS (O-antigen group B factor 4–5, Difco), α-tubulin (B5-1-2; Sigma Aldrich), α-hamster-Cy3 (Jackson), α-mouse-Cy3 (Jackson), α-mouse-Cy5 (Jackson), α-rabbit-Cy3 (Jackson), α-rabbit-Cy5 (Jackson), AlexaFluor647-conjugated wheat-germ agglutinin (Molecular Probes), TRITC-conjugated Phalloidin (Fluoprobes), AlexaFluor488 or 647-conjugated phalloidins (Molecular Probes), and DAPI (Sigma Aldrich). Automated microscopy analysis of invasion efficiency in epithelial cell lines was performed as described in detail elsewhere [77]. For confocal microscopy, we used a Zeiss Axiovert 200m microscope with 10X–100X objectives, a spinning disc module (Visitron), and two Evolve 512 EMCCD cameras (Photometrics), or a Nikon Eclipse Ti2 microscope core fitted with 10-100X objectives, a X-Light-V2-LFOV spinning disc module (Crest), and a Prime 95B 25mm camera (Photometrics). Images were captured and processed using the Visiview software (Visitron) and/or Image J ×64. For quantification of S.Tm/pssaG-GFP bacteria in mouse intestinal epithelial tissue, 20μm cross-sections stained with ICAM-1, phalloidin, and DAPI, were imaged at 400X–1,000X, and intracellular (GFP+) S.Tm were enumerated blindly in six to nine non-consecutive sections/mouse. Data represent averages/section.

Live microscopy of infected cell lines

15.000 LifeAct-expressing HeLa (LifeAct-mCherry) or MDCK (LifeAct-GFP) cells were seeded into glass-bottomed culture dishes (ibidi) in culture medium 24 hours prior to the experiment. To induce polarization of MDCK cells, 25.000 cells were seeded into culture dishes and incubated for 7 days, with medium exchange after 3, 6, and 7 days. Short-term live microscopy was performed in HBSS (Gibco)/10% FCS/20mM Hepes. Cells were infected with S.Tm^wt/pmCherry or S.Tm^wt/pGFP (MOI ~50) or with S.Tm^ΔsopBEE2/pmCherry or S.Tm^ΔsopBEE2/pGFP (MOI ~500). Movies were acquired on a Nikon Eclipse T1 inverse microscope equipped with a Yokogawa CSU-W1-T2 spinning-disk confocal unit and two EMCCD ixon888 cameras, using a 20x objective (non-polarized cells; PLAN Apochromat, NA 0.75) or a 60x oil objective (polarized MDCK; PLAN Apochromat, NA 1.4). Movies were acquired for 14 min—40 min and time-lapse data were reconstructed and analyzed in Image J ×64. Subsequent to live cell imaging, infected cells were washed with HBSS/10% FCS, fixed with 4% PFA, washed with 4% sucrose, saturated in 20% sucrose, and incubated in a PBS/3% bovine serum albumin/3% sucrose blocking buffer. Extracellular bacteria were labeled using α-S.Tm LPS and α-rabbit-Cy5. Cells were subsequently permeabilized in 0.1% Triton X-100 and stained with DAPI. Image acquisition of the same areas previously recorded by live microscopy was performed using a 100X oil objective (PLAN Apochromat, NA 1.49; non-polarized cells) or a 60X objective (PLAN Apochromat, NA 1.4; polarized MDCK cells). Image z-stacks were collected using the VisiVIEW software and further analyzed in Fiji. For consistency, the LifeAct signal is presented as red and S.Tm as green in all figure panels.

Cryo-electron microscopy and cryo-electron tomography

EM finder grids (gold NH2 R2/2, Quantifoil) were sterilized under UV light and then glow discharged. Grids were placed on the bottom of wells in a 12-well plate (Nunc, Thermo Fisher) and equilibrated in DMEM. Subsequently, 30.000 HeLa cells were seeded into each well and incubated overnight. Cells were infected with S.Tm^wt and S.Tm^ΔsopBEE2 as indicated. Plunge freezing was performed according to [78]. Briefly, grids were removed from the wells using tweezers, these subsequently mounted in a Vitrobot (Thermo Fisher) and the grids blotted from the backside by installing a Teflon sheet on one of the blotting pads. Grids were plunge-frozen in liquid ethane-propane (37%/63%) and stored in liquid nitrogen as described [79]. Infected cells were examined by cryo-electron microscopy (cryoEM) and cryo-electron tomography (cryoET) as detailed in [78]. Images were recorded on a Tecnai Polara TEM (Thermo Fisher) equipped with post-column GIF 2002 imaging filter and a K2 Summit direct electron detector (Gatan), or on a Titan Krios TEM (Thermo Fisher) equipped with a Quantum LS imaging filter and a K2 Summit. Both microscopes were operated at 300kV and the imaging filters were set to 20 eV slit width. The pixel size at the specimen level ranged between 4.29–5.95Å. Tilt series covered an angular range from -60° to +60° with 1° increments and -8 μm defocus. The total dose of a tilt series was 120 e^-/Ų. Tilt series and 2D projection images were acquired automatically using UCSF Tomo [80] or SerialEM [81]. Three-dimensional reconstructions and segmentations were generated using the IMOD program suite [82].

Field emission scanning electron microscopy

Infected mouse cecal tissue or HeLa cells were fixed in 2.5% glutaraldehyde (Polyscience). After washing in Krebs-Ringer buffer, 1% OsO₄ (Polyscience) was used for post-fixation, or samples were washed in TE buffer (TRIS 10 mM, EDTA 1 mM, pH 7.0) without further osmification. HeLa cells were additionally incubated in 0.5% carbohydrazide and treated with 1% OsO₄ for a second time. All samples were washed before dehydration in a graded series of acetone. Critical-point-drying was performed with liquid CO₂ using an Autosamdri-931 (Tousimis or Bal-Tec CPD030). Thereafter, samples were mounted on aluminum SEM stubs, sputter coated with 5nm platinum/palladium or palladium/gold (Safematic CCU-010 or Bal-Tec SCD500). Samples were examined using a Zeiss Merlin Gemini II ultra-high resolution field emission scanning electron microscope at an acceleration voltage of 5 kV. Images were captured and analyzed with Zeiss SmartSEM and Image J ×64.

Barcoded consortium infections

Barcoded S.Tm strains are detailed in S1 Table. Strains were grown in LB/0.3M NaCl/12.5μg/ml chloramphenicol for 12h at 37°C, diluted 1:20 in LB/0.3M NaCl and cultured for another 4h at 37°C. A 1:1:1:1:1:1:1 mix of all seven strains (or a 1:1:1:1:1:1 mix of a six strain consortium for S3E Fig) was used as inoculum. Cell lines were seeded in wells or atop Transwell inserts as indicated and infected with the mixed consortium inoculum for 7-20min at low MOI (i.e. 0.2–2; to limit the impact of cooperative invasion). Extracellular S.Tm were killed by 200μg/ml gentamicin for 1h and cells were washed and lysed in 0,1% sodium deoxycholate. The diluted inoculum and the retrieved intracellular bacterial population were enriched for 16h in tubes with LB (+/-12.5μg/ml chloramphenicol) at 37°C. For mouse infections, the mixed inoculum was resuspended in PBS and used to infect sm-pretreated Nlrc4^-/- mice with 5×10⁷ total CFUs per oral gavage. Animals were sacrificed ~18h p.i. The cecum was opened longitudinally, washed extensively in PBS, and treated with 400μg/ml gentamicin for 30min. The tissue was washed an additional nine times in PBS and homogenized in a TissueLyzer (Qiagen). S.Tm populations in cecum content and cecal tissue lysate were enriched in parallel in LB/12.5μg/ml chloramphenicol at 37°C. Total bacterial loads in cecum content and tissue was evaluated by plating a subfraction of the homogenates on MacConkey agar with 50μg/ml streptomycin. Genomic DNA from enrichment cultures was extracted using the GenElute^™ Bacterial Genomic DNA Kit (Sigma Aldrich) and quantitative PCR analysis performed on 9ng of total genomic DNA, using the Maxima SYBR Green/ROX qPCR Master Mix (2X) (Thermo Scientific). Primers are detailed in S2 Table. The relative abundance of each barcoded strain was normalized to the corresponding mixed inoculum for HeLa cell infections, or to the corresponding cecum content for animal infections.

Western blot

The indicated S. Tm strains were grown overnight in LB/0.3M NaCl/50μg/ml streptomycin sulphate, sub-cultured 1:20 in 3ml LB/0.3M NaCl for 4h. S.Tm were harvested, boiled in SDS-sample buffer, and particulate debris was spun down. Samples were run on 15% SDS-PAGE gels at 90V, 120min and subsequently transferred onto nitrocellulose filter (Protran BA 85, GE Healthcare). Filters were washed in PBS-Tween20 (National diagnostics) and blocked with PBS-Tween/5% milk powder. The indicated proteins were detected with primary antibodies α-M45 (purified from hybridoma), α-SipA (purified from ascites), α-σ70 (Abcam), and an HRP-conjugated rabbit anti-mouse secondary antibody. Signals were revealed with the ECL chemiluminescence detection reagent (Amersham) onto SuperRX Fuji medical x-ray film (Fujifilm corporation), using a Fujifilm FPM 800A developer.

Statistical analysis

Where applicable, statistical significance was assessed by the Mann—Whitney U-test, the Kruskal—Wallis with Dunn’s post-test, or the one-way ANOVA with Dunnett´s multiple comparisons test, as indicated in the figure legends.