The facultative intracellular pathogen Salmonella enterica remodels the host endosomal system for survival and proliferation inside host cells. Salmonella resides within the Salmonella-containing vacuole (SCV) and by Salmonella-induced fusions of host endomembranes, the SCV is connected with extensive tubular structures termed Salmonella-induced filaments (SIF). The intracellular lifestyle of Salmonella critically depends on effector proteins translocated into host cells. A subset of effectors is associated with, or integral in SCV and SIF membranes. How effectors reach their subcellular destination, and how they interact with endomembranes remodeled by Salmonella remains to be determined. We deployed self-labeling enzyme tags to label translocated effectors in living host cells, and analyzed their single molecule dynamics. Translocated effectors diffuse in membranes of SIF with mobility comparable to membrane-integral host proteins in endomembranes. Dynamics differ between various effectors investigated and is dependent on membrane architecture of SIF. In the early infection, host endosomal vesicles are associated with Salmonella effectors. Effector-positive vesicles continuously fuse with SCV and SIF membranes, providing a route of effector delivery by translocation, interaction with endosomal vesicles, and ultimately fusion with the continuum of SCV/SIF membranes. This mechanism controls membrane deformation and vesicular fusion to generate the specific intracellular niche for bacterial survival and proliferation.
Various intracellular pathogens are confined to membrane-bound compartments. Within these organelles, pathogens are able to adopt specific intracellular lifestyles. Biogenesis of specialized pathogen-containing vacuoles depends on recruitment of subsets of host cell endosomes in order to establish nutritional supply, and to evade the host immune defense. For this purpose, pathogens translocate, by different secretion systems, specific effector proteins that manipulate the host cell endosomal system1.
Salmonella enterica is a Gram-negative, foodborne bacterial pathogen, causing diseases ranging from severe typhoid fever to self-limiting gastrointestinal infections in various hosts. S. enterica serovar Typhimurium (STM) is commonly used to investigate the intracellular lifestyle of Salmonella. After invasion or phagocytic uptake, STM initiates a complex intracellular lifestyle enabling survival and proliferation within host cells. STM resides in a membrane-bound compartment termed Salmonella-containing vacuole (SCV), which acquires late endosomal markers, but does not mature to a bactericidal compartment2. Characteristic of infected cells is the formation of tubular structures connected to the SCV. Such Salmonella-induced tubules (SIT) comprise various tubular structures composed of recruited host endomembranes of various organellar origin3. The best characterized SIT are Salmonella-induced filaments (SIF), marked by lysosomal membrane glycoproteins such as LAMP14. SIF are highly dynamic in the initial phase of intracellular lifestyle. If fully developed, SIF comprise double membranes built up during development where initial SIF are single-membrane tubular compartments (leading SIF), which over time, convert into double-membrane (trailing SIF) tubular structures5. The molecular mechanisms of these pathogen-driven events of vesicle fusion and membrane deformation remain to be determined.
SIF formation and systemic virulence of STM are dependent on functions of genes within Salmonella pathogenicity island 2 (SPI2). SPI2 encodes a type III secretion system (T3SS) which enables the translocation of various effector proteins inside the host cell6. Mutant strains deficient in SPI2-T3SS are highly attenuated in systemic virulence in the mouse model of systemic infection, and show reduced intracellular replication in cell-based models7,8. We recently reported that SIF biogenesis supports intracellular lifestyle by bypassing nutritional restriction in SCV-SIF continuum by recruiting nutrients from the host endosomal system and is therefore crucial for bacterial proliferation and survival9.
Despite the large number of effector proteins translocated by the SPI2-T3SS, only a subset of these manipulates the host endosomal system and induces vesicle fusion for SCV and SIF biogenesis. These are SifA, SseF, SseG, PipB2, SseJ and SopD26. The most severe phenotype is mediated by SifA, as mutant strains defective in sifA fail to induce SIF and show loss of SCV integrity leading to bacterial release into host cytosol, and attenuation in intracellular proliferation and systemic virulence10. SseF, SseG, and PipB2 contribute to SIF formation, as mutant strains lacking the effectors show aberrant SIF biogenesis. Infection with sseF-deficient STM leads to the formation of only single-membrane SIF, and infection with pipB2-deficient strains results in the induction of enlarged bulky SIF5,11.
A subsets of SPI2-T3SS effectors is recruited to Salmonella-modified membranes (SMM) after translocation that is prominently associated with membranes of the SCV and SIF network12. This subcellular localization appears to be prerequisite for effector and host protein interactions that mediate vesicle fusion and SIF elongation13. However, the molecular mechanisms of effector targeting to SMM are poorly understood. In STM-infected cells, a dynamic extension of SIF network was observed, raising the question how SPI2-T3SS effector integrate into SMM. For example, highly hydrophobic effectors like SseF appear exclusively associated with SIF membranes14,15. As T3SS translocation delivers effector proteins into host cell cytosol, specific mechanisms of targeting and integration into host endosomal membranes are required, and we here applied novel imaging approaches to unravel these mechanisms.
We recently established an imaging approach utilizing self-labeling enzyme (SLE) tags fused to STM effector proteins to enable super-resolution microscopy (SRM), and single-molecule imaging of effector dynamics in living cells16. Here we applied these approaches to investigate the delivery of SPI2-T3SS effectors to SMM, their localization, and dynamics in SMM. This study provides new insights in the delivery mechanism of effector proteins to the SCV-SIF continuum.
Continuous interactions of SCV, SIF, and host cell endosomal compartments
We analyzed interactions of intracellular STM with the host cell endosomal system. Pulse/chase experiments with fluorochrome-conjugated gold nanoparticles (nanogold) allowed to label the lumen of endosomes17. A subset of these endosomes was in contact with dynamic SIF and events of fusion between nanogold-labeled endosomes and SIF were detected (Supplementary Movie 1). Due to the transient nature, fusions between host cell endosomes and membranes of SCV or SIF were rarely determined, and Supplementary Movie 1 shows a representative event. In contrast to other fluid tracers that become rapidly diluted after a fusion between endosomes and SIF, the aggregation of nanogold led to the formation of distinct foci that were readily detectable after fusion events. We next performed ultrastructural analyses of STM-infected cells with markers for vesicular fusions (Supplementary Fig. 1). Decoration of endosomal membranes with LAMP1-miniSOG lead to 3,3’-diaminobenzidine (DAB) conversion in endosomal lumen and deposition of DAB polymers in SIF lumen (Supplementary Fig. 1a). Pulse/chase of STM-infected cells with nanogold BSA-rhodamine labeled vesicular compartments associated and fusing with SCV and SIF (Supplementary Fig. 1b).
Interactions of endosomal compartments with SIF were frequently observed in TEM analyses of infected host cells (Supplementary Fig. 2a). Using live-cell correlative light and electron microscopy (CLEM), infected cells were imaged during the formation of dynamically extending SIF (Fig. 1, Supplementary Fig. 2b–i). Correlation identified LAMP1-positive tubular vesicles in connection with SCV harboring STM. The investigated cell showed double-membrane (dm) SIF distal to the SCV (Fig. 1e), or in connection to SCV (Fig. 1f, g). In few occasions, the contact of single-membrane (sm) vesicles of spherical appearance with dm SIF was observed (Fig. 1h, i, j). Although the temporal resolution of our live-cell imaging (LCI) approach did not allow us to distinguish fusion from fission events for the vesicle, our data would be in line with a fusion of a host cell endosome with a dm SIF and release of luminal content in the outer lumen of SIF.
Distribution of SPI2-T3SS effector proteins on SCV and SIF membranes
The previous data revealed the fusogenic properties of the SCV/SIF continuum, and indicated how the SIF network is dynamically expanding. Prior work demonstrated that the formation of the SIF network is dependent on translocated SPI2-T3SS effector proteins, and that subsets of these effector proteins are closely associated with SIF membranes15,18,19. Thus, we next followed the distribution of PipB2 as representative SPI2-T3SS effector protein over the course of STM infection (Fig. 2a). In the early phase (4 h p.i.), the signal intensity for PipB2 immunostaining was low, and most of the signals were associated with small spherical vesicles. At 8 h p.i., a SIF network was developed and PipB2 signals were mostly associated with SIF and SCV membranes. At the end of observation, i.e. 16 h p.i., PipB2 signal was strongly increased and was almost exclusively colocalized to membranes of SIF and SCV. A similar subcellular distribution over time of infection was observed for other membrane-associated SPI2-T3SS effectors such as SseF and SseJ (Supplementary Fig. 3).
To investigate if translocated effector proteins are present in membranes of endosomal compartments prior to integration of these membranes into the SCV/SIF continuum, we applied immunogold labeling for TEM analyses. A triple HA-tagged allele of sseF was used as representative membrane-integral SPI2-T3SS effector protein. SseF-3xHA was synthesized, translocated, and subcellular localized as observed for SseF-HA (Supplementary Fig. 4). For optimal preservation of endosomal membranes and epitopes we applied the Tokuyasu technique20 for immunolabeling on ultrathin hydrated sections. In infected HeLa cells, immunogold-labeled SseF-3xHA was associated with SCV membranes (Fig. 2). We also detected labeling for SseF associated with spherical membrane compartments distal to the SCV. In ultrathin sections, such signal could result from cross-sections of small spherical vesicles, or of extended tubular compartments such as SIF. To distinguish these forms, consecutive ultrathin sections were inspected, indicating labeling a single section rather than in compartments extended through several sections (Supplementary Fig. 4).
These ultrastructural observations support a model that effector proteins associate with and integrate in host cell endosomal membranes prior to incorporation into the SCV/SIF continuum.
Models for SPI2-T3SS effector targeting to endomembranes
It is not known how hydrophobic effector proteins insert into host cell endomembranes. We built several hypotheses for the route of SPI2-T3SS effector proteins from translocation to their final destination (Fig. 3). In model a, effector proteins are directly integrated into SCV membranes after translocation. In model b, effector proteins are translocated into the host cell cytosol, and a fast interaction with unknown bacterial or host cell proteins enables insertion into host endomembranes. In model C, direct delivery of effector proteins into host vesicular membranes is mediated by the SPI2-T3SS itself, and no cytosolic effector intermediates are present. In model a, peripheral distribution of effector proteins is mediated by tubulation of SCV membranes containing effector proteins. In models b and c, effector proteins are first inserted into endosomal membranes that subsequently fuse with developing SIF. We would also consider combinations of the models, and distinct modes of delivery for different effector proteins. We set out to test these models by applying a recently developed LCI approach for translocated effector proteins on single molecule level16.
SPI2-T3SS effector proteins are highly dynamic on SIF membranes
To follow the dynamics of SPI2-T3SS effector proteins on or in SIF membranes, we deployed single-molecule localization and tracking microscopy (TALM)21. As host cells, HeLa cells were used that constitutively express LAMP1-monomeric enhanced green fluorescent protein (LAMP1-GFP) to allow visualization of SCV and SIF. Host cells were infected with STM mutant strains deficient in genes for specific effectors. The strains harbored plasmids encoding effector proteins fused to HaloTag, a SLE tag, and infected cells were labeled with HaloTag ligand coupled to the fluorescent dye tetramethylrhodamine (HTL-TMR). As previously shown16, the effector proteins SseF, SifA, and PipB2 fused to HaloTag can be localized in infected host cells 8 h p.i. and a complete colocalization with LAMP1-GFP-positive SCV and SIF membranes was observed (Fig. 4a, Supplementary Fig. 5, Supplementary Movies 2, 4, 5).
We tracked the movement of the key STM effector proteins SseF, SifA, and PipB2 fused to HaloTag on Salmonella-modified membranes. By analyzing comprehensive data sets of single-molecule trajectories, the mobility of effector proteins using pooled trajectories resulting in a two-dimensional diffusion coefficient (DC), extracted from mean square displacements (MSD), was demonstrated16. As control, the host membrane protein LAMP1 was used and visualized after transient transfection of HeLa LAMP1-GFP cells for expression of LAMP1-HaloTag (Supplementary Movie 3). For non-moving particles, tracking of PipB2-HaloTag on SIF tubules was performed in fixed host cells (Supplementary Movie 6).
The DC of fixed PipB2-HaloTag was quantified as 0.009 ± 0.0008 µm2 × s−1. For LAMP1-HaloTag, a DC of 0.055 ± 0.01 µm2 × s−1 was determined. The effector proteins SifA, SseF, and PipB2 fused to HaloTag varied in their mobility with DC of 0.058 ± 0.009, 0.088 ± 0.01 and 0.11 ± 0.01 µm2 × s−1, respectively (Fig. 4b, and Supplementary Movies 2, 4, 5). These values are in line with data in our previous report describing the technique16. In all cases, the trajectories developed bidirectional, without preferential movement of molecules towards SCV-proximal or SCV-peripheral portions of SIF.
The SPI2-T3SS effector proteins investigated here all are associated with host cell endosomal membranes after translocation. For several effectors, interacting host proteins are known, such as SKIP and PLEKHM1 for SifA22,23. For SifA, the endomembrane targeting is due to prenylation of a C-terminal CxxC motif24. We investigated if the ablation of membrane binding of SifA alters dynamics in host cells. A mutant SifA allele lacking the CxxC motif (SifAΔ6-HaloTag) was translocated by intracellular STM and showed association with SIF membranes. This allowed SMT, and mean DC of 0.089 µm2 × s−1 (±0.046) was determined (Fig. 4c, Supplementary Fig. 6). The DC of SifAΔ6-HaloTag was about twice the motility of SifA-HaloTag, indicating that lack of prenylation severely increases diffusion rate.
We also mutated coiled-coil domains proposed to mediate membrane association of PipB2, SseJ, or SopD225. The mutant alleles of these effectors also showed altered motility on SIF membranes. Further studies will reveal the role of these domains in interaction with endomembranes and effects on mobility.
To test the broader physiological relevance of effector dynamics, we used an infection model with the commonly used murine macrophage cell line RAW264.7. RAW264.7 permanently transfected for LAMP1-GFP expression were used to phagocytose stationary phase STM. After adaptation to intracellular conditions, STM initiated SPI2-T3SS translocation resulting in endosomal remodeling and SIF formation. Translocated effector proteins fused to HaloTag were analyzed SMT as shown for SifA-HaloTag (Fig. 4d, Supplementary Fig. 7). The movement of SseF, PipB2 and SifA on SIF membranes was analyzed and DC were determined (Fig. 4d, Supplementary Fig. 7). The DC for SseF, SifA, and PipB2 fused to HaloTag were similar to those determined in HeLa cells. As observed for HeLa cells, in RAW264.7 the DC of SifA was lower than DC of SseF or PipB2.
Infection with STM mutant strain ΔsseF leads to increased formation of sm SIF which are smaller in diameter and volume. Fully developed SIF in STM WT-infected cells are predominantly dm SIF5,11. We analyzed SPI2-T3SS effector mobility on sm SIF to investigate potential effects of SIF architecture on the distribution and diffusion of effector proteins. HeLa LAMP1-GFP cells with STM ΔsifA ΔsseF strain expressing sifA::HaloTag and dynamics of SifA-HaloTag molecules on sm SIF were analyzed (Fig. 5a, b, Supplementary Movies 7, 8). The reduced diameters of sm SIF were verified by intensity profile analyses of accumulated SifA-HaloTag trajectories on SIF induced by STM WT and ΔsseF strains (Fig. 5d). When calculating DCs for LAMP1-HaloTag and SifA-HaloTag on sm SIF induced in cells infected by STM ΔsseF, a reduction of mobility with DC values of 0.028 ± 0.008 and 0.035 ± 0.004 µm2 × s−1, respectively was observed (Fig. 5c).
Taken together, these single-molecule analyses demonstrate that SPI2-T3SS effector proteins are highly dynamic on SIF. PipB2 showed distinct higher mobility in comparison to host membrane-integral protein LAMP1. The mobility of SifA and LAMP1 was reduced on sm SIF in comparison to dm SIF.
SPI2-T3SS effectors accumulate on leading SIF during transition to trailing SIF
After succeeding in imaging effectors on sm SIF, we analyzed the transition of leading to trailing SIF. Thinner leading SIF consists of single-membrane tubules and the connected trailing SIF consist of double-membrane structures, i.e. fully developed SIF. It was proposed that this transition is facilitated by a lateral extension of membranes of leading SIF, engulfment of host cell cytosol and cytoskeletal filaments, and finally membrane fusion to form double-membrane trailing SIF5. To image the transition from leading to trailing SIF, translocated SseF-HaloTag was analyzed by LCI at 6 h p.i. In Fig. 5e and Supplementary Movie 9, a thin SIF with a weak GFP signal was imaged, and a wider trailing SIF with strong GFP signal was developing alongside the thinner structure. By collecting and localizing all signals of SseF-HaloTag between the frames of the growing LAMP1-positive SIF tubule, increased concentration of effector proteins on the leading SIF before transition to trailing SIF became apparent. This was also shown for SifA-HaloTag and PipB2-HaloTag (Supplementary Fig. 8a, b, Supplementary Movies 10, 11). These effector proteins were also localized at leading SIF when transforming to trailing SIF, however the local concentration was less pronounced.
These findings indicate that SPI2-T3SS effector proteins are already present on leading SIF, and in particular SseF appears to be involved in the transformation to trailing dm SIF, as an accumulation of effector protein can be detected directly before transition.
Effector proteins target endosomal vesicles in the early phase of infection
The presence and concentration of effector proteins on leading SIF suggest a delivery mechanism of effectors to the tips of growing sm SIF tubules. To address the question how SPI2-T3SS effector proteins reach their subcellular destination in an infected host cell, we applied LCI by confocal laser-scanning microscopy (CLSM) of infected HeLa LAMP1-GFP cells at 4 h p.i. In the early stage of infection, the SCV is already formed, while SIF biogenesis initiates. We found that after labeling of effector proteins, also bacteria were heavily stained, indicating large amounts of effector proteins stored in bacteria. By monitoring the HaloTag-fused PipB2, SseF, SseJ, and SteC, localization of effector proteins in a punctate, vesicle-like manner was observed in infected cells. These structures showed most frequently colocalization with LAMP1-GFP signal, but also labeled endomembrane compartments lacking the late endosomal marker were observed (Supplementary Fig. 9).
As prior work indicated the interaction of SseF and SseG26, and function of both effector proteins is required for formation of dm SIF and efficient intracellular proliferation5,9, we followed the translocation and potential interactions of SseF and SseG (Supplementary Fig. 10a). Immunolabeling of epitope-tagged SseF and SseG indicated differential targeting to LAMP1-positive endosome. Quantification indicated SseF colocalization with LAMP1 already in the early phase of 4 h p.i., while SseG colocalization with LAMP1 was delayed and at lower levels (Supplementary Fig. 10b). The colocalization of SseF and SseG increased over time and colocalized effector were predominantly located on SIF (Supplementary Fig. 10a). To further analyze SseF and SseG interaction, we applied SMT of SseF-HaloTag in the background of STM strains either lacking SseF or both SseF and SseG (Supplementary Fig. 10c). The diffusion coefficient for SseF-HaloTag was increased in STM ΔsseF background compared to STM WT, and slightly lower in background of STM ΔsseFG. These data indicate the spatial interaction of SseF and SseG during endosomal remodeling and suggest that the proper balance in the amounts of the translocated effectors affect their interaction.
SifA-HaloTag was not detected decorating vesicles in the early stage of infection. The low level of SifA-HaloTag translocation could hamper visualization. These findings are in line with the observation made by SRM localization with SifA-HaloTag showing the lowest effector concentration, while PipB2-HaloTag showed the highest labeling intensity on SIF tubules. Accordingly, vesicles marked with PipB2-HaloTag could be easily imaged in the early stage of infection, and PipB2 protein was chosen for further analyses. We set out to determine different phenotypes of PipB2-HaloTag localization and therefore studied 100 cells containing PipB2-HaloTag-positive vesicles.
Of note, at 4 h p.i. effector-positive vesicles were found in a subset of infected cells. We conclude that due to heterogeneity in SPI2 induction, infected cells with bacteria with low levels of effector secretion showed no detectable HaloTag signal. In line with this observation, PipB2-HaloTag fluorescence intensity on SIF at 16 h p.i. also varied between infected cells (Supplementary Fig. 11). In the early phase of infection, distinct phenotypes of PipB2-HaloTag localization can be distinguished for effector-positive vesicular structures. Moreover, the infected cells either showed no SIT, or already developing first SIT structures. The tubular structures were either LAMP1-GFP-positive, or lacking the endosomal marker, and in one population of cells these structures had already acquired PipB2-HaloTag, and others were still lacking the effector (Fig. 6a). At 4 h p.i., in total 62% of the infected cells still did not show SIF formation, yet were positive for vesicles decorated with PipB2-HaloTag. All other cells also displayed vesicles positive for PipB2-HaloTag and already formed SIT (Fig. 6b). To test the characteristics of effector-decorated vesicles in infected cells, we performed tracking analyses of vesicles. Single LAMP1-GFP-positive or PipB2-HaloTag-positive vesicles were tracked in 3D in infected cells. In co-motion tracking analyses, we observed that vesicles positive for LAMP1-GFP and PipB2-HaloTag were tracked in parallel, and the patterns of movement were identical (Fig. 7a, b, Supplementary Movie 12). When studying individual vesicle tracks, as control conditions LAMP1-GFP-decorated vesicles in non-infected cells, either nocodazole-treated or non-treated were tracked (Supplementary Fig. 12, Supplementary Movies 13, 14).
We quantified the mean track displacement length (MTDL) and the mean track speed (MTS) of pooled trajectories. In infected cells, PipB2-HaloTag-marked and LAMP1-GFP-marked vesicles did not differ in MTDL and MTS and therefore showed normal characteristics of vesicle movement. In cells treated with nocodazole, both values were significantly decreased due to the inhibition of vesicle trafficking after microtubule disruption. Interestingly, late endosomal vesicles tracked in non-infected and non-treated cells showed a more rapid movement, and MTDL and MTS values were significantly increased (Fig. 7c). These data demonstrate that SPI2-T3SS effectors are recruited to endomembrane compartments, and as analyzed in detail for PipB2-HaloTag, in infected cells in the early phase of infection PipB2-HaloTag-positive vesicles behave similar to LAMP1-positive vesicles.
PipB2-HaloTag-positive vesicles continuously integrate into the SIF network
After establishing that SPI2-T3SS effector proteins are recruited to vesicles in the early infection during SIF biogenesis, we hypothesized that delivery to SCV and SIF tubules may occur by fusion of endosomal vesicles with membrane-integrated effector proteins. We set out to study PipB2-HaloTag localization from early to late stage of infection and applied long-term LCI of infected cells. Over time, we observed a reduction of effector-decorated vesicles and extension of effector-positive SIF network (Fig. 8a, Supplementary Movie 15). Rendering the PipB2-HaloTag-labeled endomembranes, 29 effector-positive vesicle-like objects were detected 5 h p.i. while only six objects remained at 12 h p.i. Concurrent with the decreasing number of PipB2-HaloTag-decorated objects, PipB2-HaloTag-positive SIF developed (Fig. 8c), suggesting that effector-positive SIF membranes emerge from vesicles. We speculate that STM mutant strains without the ability to induce tubulation of SIF but still possessing a functional SCV should accumulate effector-positive vesicles over time. We used STM deficient in sifA and sseJ, previously reported to maintain SCV membrane but lacking SIF formation27. In infected HeLa LAMP1-GFP cells at 16 h p.i. an accumulation of PipB2-HaloTag-positive vesicles was observed, as well as lack of SIF network formation (Supplementary Fig. 11). These findings indicate that STM SPI2-T3SS effector proteins are integrated into the SCV-SIF continuum via fusion of effector-positive endomembrane compartments.
Effector recruitment to endomembrane compartments is dependent on vesicle movement
To investigate if presence of effector-decorated vesicles is dependent on dynamics of endosomal compartments, we monitored localization of PipB2-HaloTag over time in infected cells after inhibition of vesicle trafficking. For this purpose, infected cells were treated with nocodazole 2 h p.i., a time point where STM resides in SCV and activates expression of SPI2-T3SS genes28. Addition of nocodazole abrogates vesicle movement due to interference with microtubule integrity. We did not observe PipB2-HaloTag-positive vesicles in nocodazole-treated cells, implicating that vesicle movement is required for effector recruitment Fig. 8b, Supplementary Movies 16).
To address reversibility of inhibition, we imaged infected cells for 8 h after removing nocodazole by washing cells twice at 4 h p.i. A slow initiation of vesicle movement was monitored over time. Starting 11 h p.i., the first PipB2-HaloTag-decorated LAMP1-GFP-positive vesicles were imaged (Fig. 8b). Association of effector proteins with endosomal compartments was dependent on vesicle movement on microtubules, but did not require SIF formation.
We previous demonstrated that SPI2-T3SS effector proteins, as well as the host cell protein LAMP1 can be tracked, on single molecule level, on SIF and show a rapid, bidirectional movement16. Here were applied SMT to follow the fate translocated effector proteins in infected host cells. The ability of integral membrane proteins to rapidly interchange in SIF membranes has already been demonstrated by FRAP experiments and that revealed recovery of LAMP1 after photobleaching on distal SIF9. Using the split-GFP approach, PipB2 recovery on tubules was detected upon photobleaching, indicating a rapid distribution of PipB229. The diffusion coefficients (DC) are similar to values determined for mitochondrial proteins and for cytokine receptors on the plasma membrane30,31.
Interestingly, the mobility of effectors differs with PipB2 being the most mobile effector protein. Mobility of membrane proteins depends on membrane integration, membrane composition and interaction with other proteins. The association of PipB2 to lipid rafts has been demonstrated18. These dense membrane patches would presume an impaired mobility for membrane-integral proteins. Contrary, it is established that raft association is not the dominant factor in determining the overall mobility of a particular protein as different lipid raft associated proteins showed diverse DC32. Besides this, our experiments did not reveal a specific localization of PipB2 to distinct regions of SIF membranes.
Correlation of different forms of protein association with membranes to the corresponding diffusion mobility revealed that DC of prenylated proteins were higher than DC of transmembrane proteins32. We did not observe such a correlation for STM effector proteins, with SifA being the only prenylated effector protein tested33, and propose that mobility of effectors might be influenced to a large extend by their interaction with host proteins. SseF interacts with STM effector SseG and in combination with the Golgi network-associated protein ACBD3, forming a complex tethering the SCV to the Golgi34. SifA has various interaction partners inside the host cell, such as PLEKHM2 (also called SKIP) and PLEKHM122,23. The SifA complex is able to activate kinesin-1 upon binding, resulting in budding and anterograde tubulation of SIF membranes along microtubules3,35,36. PipB2 is responsible of the recruitment of auto-inhibited kinesin-1 to the vacuole membrane37. The distinct DC of effector proteins SifA, SseF and PipB2 likely indicates the form of interaction with cognate host cell molecules. Both, SifA and SseF, take part in tethering SCV and SIF to host structures, possibly resulting in reduced mobility. Hence, the molecular mechanisms behind effector mobility on SIF membrane remain to be elucidated.
We demonstrate here that effector motilities are affected by SIF architecture. The reduced DC of STM effector protein SifA, as well as host protein LAMP1 reveals slower movement on sm SIF. This indicates an overall impediment of mobility of membrane proteins in the membrane of sm SIF. This may be due to the lower membrane area of sm SIF that limits diffusion of membrane proteins perpendicular to the longitudinal axis of SIF. The smaller diameter of sm SIF compared to dm SIF leads to higher membrane curvature of sm SIF, and this may affect motility of membrane proteins. Furthermore, host cell proteins sensing membrane curvature will be recruited differentially to sm SIF and dm SIF depending on their curvature, and these proteins could have distinct effects on motilities of the proteins analyzed.
When monitoring growing SIF, we found thinner leading SIF at the extending tips, followed by development of thicker trailing SIF. Prior ultrastructural analysis revealed that leading SIF are single-membrane tubules, while trailing SIF comprise double-membrane structure, representing the fully developed SIF. It was proposed that effector protein SseF together with its interaction partner SseG are involved in conversion of sm SIF to dm SIF5. Here we present, to our knowledge first, microscopy-based evidence that effector protein SseF is present on the membranes of leading sm SIF and that the effector accumulates before engulfment to trailing dm SIF. As this is also true for SifA and PipB2, more effector proteins might be involved in the process of sm SIF to dm SIF conversion.
The concentration of effectors on the tips of growing SIF led to the question how STM effectors are able to accumulate and how they are recruited after secretion into the host cytosol. Especially SseF which is characterized by large hydrophobic domains14, could be mistargeted to cytoplasmic membranes or be prone to aggregation in host cytosol. To test models for the delivery of effector proteins to SCV-SIF continuum introduced in Fig. 3, we performed LCI with labeled effectors in the early infection. We found that various SPI2-T3SS effectors decorate host endomembranes in infected cells. The association of PipB2 with vesicles in the periphery of infected host cells has already been demonstrated18. Over time, the presence of PipB2-positive vesicles declined, corresponding with the formation of PipB2-positive SIF. These findings strengthen the hypothesis of delivery of effector proteins to SCV and SIF via fusion of effector-containing endomembranes as depicted in models b and c of Fig. 3.
The targeting of bacterial effector proteins to phagosomes and various organelles in infected host cells has been widely demonstrated38, but only few studies observed effector protein presence on host endomembranes and connected a trafficking mechanism of effector proteins to these phenomena. The Shigella effector protein IpgB1 was found to localize to endocytic vesicles in mammalian cells expressing eGFP-tagged IpgB1. Vesicles decorated with IpgB1 were found to be functional in host cell trafficking and a relocation of effector proteins from endocytic vesicles to membrane ruffles produced by Shigella was observed, indicating a delivery of effector via vesicle fusion39. Another study uses the split-GFP approach to monitor the delivery of effector AvrB by Pseudomonas via the T3SS to infected plant cells. In infected host cells, AvrB localizes to the plasma membrane, but at different time points after inoculation the localization changed to unknown vesicles, suggesting a potential trafficking of AvrB on vesicles40. Relocation of bacterial effector proteins on host endocytic vesicles to the required site of action in infected host cells might represent a universal delivery mechanism.
To further address the question how effector proteins reach their subcellular destination on host endomembranes, we analyzed effector protein localization in infected cells with inhibited vesicle movement. We found that endomembranes did not acquire PipB2 when vesicle movement was inhibited. This finding supports an insertion route of effector proteins to membranes that requires vesicle movement. In this case, effector proteins that do not require a modification by host proteins are inserted into vesicle membranes in close proximity to the T3SS, either by host chaperones, or solely determined by the sequence of membrane-integral effectors (Fig, 3, model c).
However, these hypotheses can only be applied to effector proteins without the requirement for host cell modification. SPI2-T3SS effector proteins can gain membrane association either by defined transmembrane domains within their sequence, or by host cell modifications. The effector proteins SseF and SseG are examples for the former, as these proteins contain identified transmembrane domains8,41,42. Various other SPI2-T3SS effector proteins acquire membrane association by host cell modifications. These modifications comprise different mechanisms to increase the overall hydrophobicity of effector proteins. SifA is targeted by host modifications, resulting in S-prenylation of the effector protein33. Additionally, the effector proteins SspH2 and SseI of STM have been shown to be palmitoylated to gain membrane association43. As the mentioned models do not include effectors requiring post-translational modification by the host, other mechanisms must contribute to effector relocation (Fig. 3, model d). Accordingly, we did not detect SifA on host endomembranes in infected cells.
Our data lead to various new questions. (i) During the intracellular life of STM, host endosomal compartments are gradually depleted due to fusion to SCV-SIF continuum44. Does this terminate the delivery of effector proteins and end intracellular proliferation? (ii) By which mechanism are post-translationally modified effector proteins relocated? iii) Are endomembranes specifically targeted to the T3SS, or is insertion simply dependent on proximity? Further LCI and single molecule-based analyses of STM effectors will likely contribute to answer these questions.
Bacterial strains and culture conditions
Infection experiments were performed using Salmonella enterica serovar Typhimurium (STM) NCTC 12023 strain as WT and isogenic mutant strains (Table 1). Mutagenesis was carried out as described elsewhere45. In short, strains were constructed using λ Red-mediated mutagenesis and the resistance cassette was removed using FLP-mediated recombination. Mutant strains deficient in effector genes harboring plasmids encoding the corresponding effector fused to HaloTag (Table 2) were used for microscopic analysis.
Generation of vectors for effector protein-SLE fusions and mutant alleles
Plasmids were constructed as described previously16 using oligonucleotides listed in Supplementary Table 1. Bacterial strains were cultured in Luria-Bertani broth (LB) containing 50 µg ml−1 Carbenicillin (Roth, 6344.3).
For generation of a plasmid encoding triple HA-tagged SseF, p2643 (sscB sseF::HA) was used and sseF::HA on p2643 was replaced by sseF::3HA using Gibson assembly GA. Primers for generation of vector fragment, check primers and sequence of synthetic sseF::3xHA (gBlocks, IDT) are listed in Supplementary Table 1.
Mutagenesis was performed for plasmids p4286, p4295, p4300, and p4305 encoding sseJ::HaloTag, pipB2::HaloTag, sopD2::HaloTag or sifA::HaloTag, respectively, using primers listed in Supplementary Table 1. Deletion of codons 331-336 in sifA removes the previously described prenylation site24 and was expected to reduce membrane interaction. Mutations Y281D Y284D K288D (sopD2), N309D L312D V316D (sseJ), and Y54D L57D M61D (pipB2) affect coiled-coil (CC) domains that were reported to contribute to membrane interactions of translocated T3SS effector proteins25. Site-directed mutagenesis was performed using the Q5 SDM kit (NEB, E0554S), and resulting plasmids were confirmed by sequencing (SeqLab, Göttingen).
Culture of eukaryotic cells
The non-polarized epithelial HeLa cell line (ATCC no. CCL-2) stably transfected with LAMP1-GFP5 was cultured in Dulbecco´s modified Eagle´s medium (DMEM) containing 4.5 g × l−1, glucose, 4 mM stable glutamine and sodium pyruvate (Merck Sigma-Aldrich, D6429) and supplemented with 10% inactivated fetal calf serum (Thermo, Gibco, 10270). Cells were maintained in a cell culture incubator (37 °C, 5% CO2). Murine macrophage-like cell line RAW264.7 stably transfected with LAMP1-GFP cells was cultured as described before5.
Host cell infection
HeLa LAMP1-GFP cells were seeded in surface-treated eight-well plates ((Ibidi, 80827) or on 24 mm diameter coverslips (VWR, Marienfeld 0117640) in six-well plates (Faust, TPP92006). For infection experiments, cells were grown to 80% confluence (eight-well: ~8 × 104, six-well: ~6 × 105). STM strains were grown overnight in LB supplemented with 50 µg ml−1 carbenicillin and subcultured 1:31 in fresh LB for 3.5 h. Cells were infected at MOI 50 or 75 for 25 min, washed thrice with phosphate-buffered saline (PBS), and incubated with DMEM containing 100 µg ml−1 gentamicin (Applichem, A1492) to kill non-internalized bacteria. Subsequently, medium was replaced by DMEM containing 10 µg ml−1 gentamicin for the rest of the experiment.
RAW264.7 LAMP1-GFP cells were seeded in surface-treated 8-well plates. For infection experiments, cells were grown to 80% confluence (per well ~1.6 × 105 cells). RAW264.7 cells were activated with 5 ng ml−1 recombinant murine Interferon-γ (R&D system, 485-MI) 1-day prior infection. For infection of RAW264.7 cells, STM strains were grown overnight in LB supplemented with 50 µg ml−1 carbenicillin. Cells were infected at MOI 50 for 25 min, washed thrice with phosphate-buffered saline (PBS), and incubated with DMEM containing 100 µg ml−1 gentamicin to kill non-internalized bacteria. Subsequently, the medium was replaced by DMEM containing 10 µg ml−1 gentamicin for the rest of the experiment (11 h).
Pulse-chase with gold nanoparticles
Pulse-chase labeling of the endosomal system with custom-made rhodamine-conjugated gold nanoparticles was performed as previously described17.
Correlative light and electron microscopy (CLEM)
CLEM of HeLa LAMP1-GFP cells infected by STM WT was performed as previously described5. Briefly, HeLa LAMP1-GFP cells were grown on MatTek dishes with a gridded coverslip (MatTek P35G-1.5-14-CGRD-D) and infected with the respective STM strains at MOI 75. Cells were fixed with 2% paraformaldehyde (PFA, Sigma-Aldrich, 158127) and 0.2% glutaraldehyde (GA, Sigma-Aldrich 111-30-8) in 0.2 M HEPES (Sigma-Aldrich, H7006) for 30 min prior to LM. After rinsing the cells for three times with 0.2 M HEPES buffer, unreacted aldehydes were blocked by incubation with 50 mM glycine in buffer for 15 min, followed by rinses in buffer. CLSM was performed and ROIs were chosen. Afterwards cells were fixed with 2.5% glutaraldehyde and 5 mM CaCl2 in 0.2 M HEPES in preparation for TEM. Further steps including post-fixation, dehydration, sectioning and imaging in the TEM were conducted as previously described5.
Live-cell super-resolution localization and tracking microscopy
Localization and tracking of single molecules were done as previously described16. Briefly, infected HeLa LAMP1-GFP or RAW264.7 cells were labeled with the HaloTag ligand coupled to tetramethylrhodamine (HTL-TMR, Promega G8251) in a concentration of 20 nM for 15 min at 37 °C. After 10 washing steps, the cells were imaged with an imaging medium consisting of Minimal Essential Medium (MEM) with Earle’s salts, without NaHCO3, without L-Glutamine, without phenol red (Merck D1145) and supplemented with 30 mM HEPES, pH 7.4. TIRF microscopy was performed using an inverted microscope Olympus IX-81, equipped with an incubation chamber maintaining 37 °C and humidity, a motorized 4-line TIRF condenser, a ×150 objective (UAPON 150x TIRF, NA 1.45), a TIRF quadband polychroic mirror (zt405/488/561/640rpc), a 488 nm laser (150 mW, Olympus), and a 561 nm laser (150 mW, Olympus). Localization, as well as tracking of single molecules, were carried out with the help of a self-written user interface in MatLab 2013a21,46,47,48,49,50.
Live-cell fluorescence microscopy
Infected HeLa LAMP1-GFP cells were labeled with HTL-TMR with a final concentration of 1 µM for 30 min directly before LCI. Cells were washed thrice with PBS and the media was replaced with imaging media as described above. For inhibition of vesicle movement cells were incubated with 5 µg ml−1 nocodazole (Sigma-Aldrich, M1404) 2 h p.i. Directly before LCI, cells were washed twice to remove nocodazole and labeled as described above. Imaging was done using either the confocal laser-scanning microscope Leica SP5 (Leica) equipped with an incubation chamber, a ×100 objective (HCX PL APO CS, NA 1.4-0.7) and a polychroic mirror (TD 488/543/633) or the Cell Observer Spinning Disk microscope (SDM, Zeiss) equipped with a Yokogawa Spinning Disc Unit CSU-X1a 5000, an incubation chamber, ×63 objective (α-Plan-Apochromat, NA 1.4), two EMCCD cameras (Evolve, Photometrics) and the filter combination GFP with BP 525/50 and RFP with BP 593/46 for dual cam imaging. Images were acquired and processed using the corresponding software LAS AF (Leica) and ZEN (Zeiss).
Image analysis by Imaris
Vesicle tracking and surface analysis were analyzed by Imaris 9.2.1 software package (Bitplane). Surface analysis was done to determine the amounts of effector-positive vesicles inside an infected cell. By the surface tool, data acquired by LCI were analyzed in the red (PipB2-HaloTag-TMR) channel. Vesicle volume was adjusted using auto-threshold and smoothing of 0.1, and SIF volume was adjusted using auto-threshold and smoothing of 0.3. For vesicle tracking, the spot tool was used. Spot detection was performed with the following parameters XY diameter: 0.75 µm, active model PSF elongation: 1.5 µm, and background subtraction. For tracking the autoregressive motion algorithm was chosen with a maximum gap size of 3.
Image analysis by FIJI
The FIJI package51 was used to determine intensity profiles of accumulated SifA-HaloTag trajectories on SIF. Using the line tool and subsequently, the plot profile tool, the intensity profile of the trajectories labeling the SIF was calculated and the relative distances in pixel were compared.
Statistics and reproducibility
Each experiment was repeated at least twice with similar results, using independent experimental samples and statistical tests as specified in the figure legends. Source data with sample sizes, number of technical and/or biological replicates, means, standard deviations, and calculated p values (where applicable) are provided in the Source Data file for Figures and Supplementary Figures.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Source data are provided with this paper. The microscopy data generated in this study have been deposited in the OsnaData repository under accession code: https://doi.org/10.26249/FK2/DC45CV. All plasmids described in this work are available at Addgene (Addgene IDs 198142-198151) Source data are provided with this paper.
The code for the SlimFast application is available under: https://github.com/CPaoloR/SLIMfast.
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This work was supported by the DFG through grants HE 1964/18-2, SFB 994 project Z, and SFB 1557 project 8 to M.H. We like to thank Jacob Piehler (Div. Biophysics), Christian Richter (Div. Biophysics), Rainer Kurre (iBiOs), and Michael Holtmannspötter (iBiOs) for continuous support and fruitful discussions. The support by Susanne Kunis in data management is kindly acknowledged.
Open Access funding enabled and organized by Projekt DEAL.
The authors declare no competing interests.
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Göser, V., Sander, N., Schulte, M. et al. Single molecule analyses reveal dynamics of Salmonella translocated effector proteins in host cell endomembranes. Nat Commun 14, 1240 (2023). https://doi.org/10.1038/s41467-023-36758-9
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