Cellular Microbiology (2015) 17(5), 639–647

doi:10.1111/cmi.12441 First published online 10 April 2015

Microreview Take the tube: remodelling of the endosomal system by intracellular Salmonella enterica Viktoria Liss and Michael Hensel* Abteilung Mikrobiologie, Fachbereich Biologie/Chemie, Universität Osnabrück, Barbarastr. 11, Osnabrück 49076, Germany. Summary Salmonella enterica is a facultative intracellular pathogen residing in a unique host cell-derived membrane compartment, termed Salmonellacontaining vacuole or SCV. By the activity of effector proteins translocated by the SPI2-endoced type III secretion system (T3SS), the biogenesis of the SCV is manipulated to generate a habitat permissive for intracellular proliferation. By taking control of the host cell vesicle fusion machinery, intracellular Salmonella creates an extensive interconnected system of tubular membranes arising from vesicles of various origins, collectively termed Salmonella-induced tubules (SIT). Recent work investigated the dynamic properties of these manipulations. New host cell targets of SPI2-T3SS effector proteins were identified. By applying combinations of live cell imaging and ultrastructural analyses, the detailed organization of membrane compartments inhabited and modified by intracellular Salmonella is now available. These studies provided unexpected new details on the intracellular environments of Salmonella. For example, one kind of SIT, the LAMP1-positive Salmonellainduced filaments (SIF), are composed of doublemembrane tubules, with an inner lumen containing host cell cytosol and cytoskeletal filaments, and an outer lumen containing endocytosed cargo. The novel findings call for new models for the biogenesis of SCV and SIT and give raise to many open questions we discuss in this review.

Received 27 January, 2015; revised 10 March, 2015; accepted 19 March, 2015. *For correspondence. E-mail michael.hensel @biologie.uni-osnabrueck.de; Tel. +49 (0)541 969 3940; Fax +49 (0)541 969 3942.

Introduction Salmonella enterica is a food-borne Gram-negative pathogen causing a high number of infectious diseases ranging from localized, self-limiting gastroenteritis to systemic, life-threatening typhoid fever. Salmonella is an invasive, facultative intracellular pathogen residing in a unique membrane-bound compartment, termed Salmonellacontaining vacuole or SCV (Haraga et al., 2008). Salmonella within the SCV deploys the Salmonella pathogenicity island 2-encoded type III secretion system (SPI2-T3SS) to translocate effector proteins that manipulate various host cell functions, including the vesicular transport and the organization of the endosomal system (reviewed in Ibarra and Steele-Mortimer, 2009, Rajashekar and Hensel, 2011, Figueira and Holden, 2012). The ability of Salmonella to survive within eukaryotic host cells is closely linked to systemic pathogenesis. Mutant strains defective in intracellular survival and replication such as SPI2-T3SSdeficient strains are highly attenuated in systemic disease models of infection (Hensel et al., 1998). This review will summarize recent studies of the intracellular lifestyle of Salmonella in mammalian cells, introduce novel hypotheses for the manipulation of the host cell endosomal system by Salmonella, and raise several open questions to be addressed by future work. SCV – the intracellular habitat of Salmonella SCV is considered to be a unique pathogen-containing compartment that is detoured from the canonical pathway of endosomal maturation. This altered maturation route results in generation of a specialized compartment with certain characteristics of late endosomes, while being permissive for intracellular survival and replication of Salmonella. Endosomal maturation markers such as the late endosomal/lysosomal glycoprotein (lgp) LAMP1 and the small GTPase Rab7 are present on the SCV. The SCV lumen is considered acidic with a pH of around 5. Although late endosomal/lysosomal organelle markers are present on the SCV, the activity of lysosomal hydrolases inside the SCV appears to be low. One dramatic consequence of the pathogenic interference

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cellular microbiology

640 V. Liss and M. Hensel Table 1. Types of SIT Acronym

Name

Key marker

Appearance (p.i.)

SIF SIST LNT SNX3 tubule SVAT

Salmonella-induced filaments Salmonella-induced SCAMP3 tubules LAMP1-negative tubules Sorting nexin 3 tubules Spacious vacuole-associated tubules

lgp SCAMP3 SPI2-T3SS effectors SNX3 SNX1

3–16 h 8–14 h 3–16 h 10–60 min 10–60 min

mediated by intracellular Salmonella is the induction of complex networks of tubular membrane compartments, such as Salmonella-induced filaments (SIF) that are characterized by the presence of lgp such as LAMP1 (Garcia-del Portillo et al., 1993). Recently, further Salmonella-induced tubular compartments have been described in addition to SIF (reviewed in Schroeder et al., 2011), termed Salmonella-induced SCAMP3 tubules (SIST, Mota et al., 2009) and LAMP1-negative tubules (LNT) (Schroeder et al., 2010). In addition, for early time points of Salmonella infection, further induced tubules i.e. the spacious vacuole-associated tubules (SVAT) and sorting nexin 3 tubules (SNX3 tubules) were observed (Bujny et al., 2008; Braun et al., 2010). In the following, we will use the terms Salmonella-induced tubules (SIT) if no distinction between SIF, SIST, LNT, SVAT or SNX3 tubules is possible (see Table 1), and Salmonellamodified membranes (SMM) to summarize all host cell membranes modified by activities of intracellular Salmonella.

SPI2-T3SS – the key factor for Salmonella intracellular lifestyle SPI2-T3SS activity is of central importance for the intracellular lifestyle of Salmonella and for systemic virulence in animal models of infection. This system is activated in intracellular Salmonella and translocates more than 30 effector proteins across the SCV membrane, which collectively results in intracellular survival and proliferation of Salmonella. Despite the large number of effector proteins identified so far, only a subset may be involved in direct manipulation of the intracellular habitat for induction of SIT. SifA is an important effector, as sifA mutant strains are most highly attenuated in systemic virulence (Beuzon et al., 2000). A SifA-deficient strain lacks SIF (Stein et al., 1996) and other SIT, is defective in maintaining an intact SCV, and shows an increased release into the cytosol (Beuzon et al., 2000). Other effector subsets may have roles in modification of immune responses, interference with cell migration, interference with ubiquitination, control of apoptosis and many others aspects of host–pathogen interaction (reviewed in Figueira and Holden, 2012, Pilar et al., 2013).

The intracellular habitat of S. enterica In contrast to many other intracellular pathogens that inhabit a single vacuole containing the growing population of bacteria, Salmonella maintains SCV that are tightly enclosing individual bacterial cells (Eswarappa et al., 2010). During intracellular replication, the continuous extension of the SCV surface is required to maintain this situation. However, it is not known why the intracellular lifestyle of Salmonella demands individual tight SCV. Furthermore, a reduction in acidic lysosomes was observed in parallel to the increase in SCV number. The intracellular activity of Salmonella results in the generation of a large interconnected network of tubular filaments. SIF were the first SIT identified (Garcia-del Portillo et al., 1993) and later shown to be induced upon translocation of SPI2-T3SS effector proteins (Stein et al., 1996; Beuzon et al., 2000). For the formation of SIF, intracellular Salmonella induces the conversion of the host cell endosomal system into a complex interconnected network of LAMP1-positive tubules resulting in a strong decrease in the number of normal spherical late endosomal/lysosomal vesicles and the concomitant increase in tubular compartments, assuming a steady state of the amount of lgp (Rajashekar et al., 2008). Although the cellular origin and physiological role of most types of SIT is only partially known, their presence indicates that intracellular Salmonella induce fusion and tubulation of various types of endosomes and other host cell vesicles in a rather broad manner.

Recent advances in the understanding of the intracellular habitat of Salmonella SCV and SIF are accessible to various types of endocytic cargo. Pulse/chase experiments with fluid phase markers are commonly used to follow the cellular processing of endocytosed material. Applying these techniques to Salmonella-infected cells, it was shown that endocytosed material can enter the SCV lumen as well as the SIF network (Drecktrah et al., 2008; Zhang and Hensel, 2013; Krieger et al., 2014). Live cell imaging also revealed that fluid phase makers applied at various stages prior to, or after infection reach the SIF and SCV and appear rapidly © 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 639–647

Intracellular Salmonella distributed within an interconnected network. We consider this finding as very important for the understanding of the intracellular physiology of Salmonella. The continuous fusion and delivery of various types of endocytic cargo may provide the bacteria with nutrients required for the life in the otherwise nutrient-poor SCV. Induction of endosomal tubulation was found in various epithelial cell lines, as well as in phagocytic cells (Rajashekar et al., 2008; Krieger et al., 2014). Interestingly, phagocytic cells show also without an infection large amounts of highly dynamic tubular endosomes (Knapp and Swanson, 1990). However, Salmonella infection of phagocytic cells resulted in the induction of an additional network of tubular endosomes and the formation of double-membrane SIF similar to those observed in epithelial host cells (Krieger et al., 2014). Proteomics of SCV and SIT reveal interaction with various host cells compartments. In order to understand the intracellular lifestyle of Salmonella in mammalian host cells, the characterization of the specific properties of the SCV is a key issue. While canonical organelle marker proteins have been analysed for characterization of SCV biogenesis, a systematic proteomic inventory of the membrane proteomes of SCV and SIT may provide important clues to the biogenesis of the pathogencontaining compartment, as recently exemplified for Legionella pneumophila (Urwyler et al., 2009). By applying quantitative proteomics, Auweter et al. (2011) identified novel host cell interaction partners for various SPI2-T3SS effector proteins, such as desmosome proteins for SseF/SseG, and Talin and OSBP for SseL. To explore the proteome composition of the membrane environment of intracellular Salmonella, an enrichment and proteomics of SMM was performed (Vorwerk et al., 2015). The analysis revealed for SMM the presence of proteins indicative for trans-Golgi network (TGN), recycling endosomes, endoplasmic reticulum and further host organelles. This suggests that membranes of rather heterogeneous origin become associated with SCV and SIT. Although proteome analyses are subjects to contamination with host cell organelles, these findings are in line with previous observations of manipulation of cellular trafficking (e.g. see Salcedo and Holden, 2003, Kuhle et al., 2006, Drecktrah et al., 2007, Zhang and Hensel, 2013) and indicate that intracellular Salmonella hijack host cell compartments for fusion to the SCV and SIT network with rather low selectivity. Salmonella within the SCV bypass endosomal maturation. A key SPI2-T3SS effector protein for the intracellular lifestyle of Salmonella is SifA (Beuzon et al., 2000) and multiple functions have been assigned to SifA. SifA interacts with host cell protein SKIP (Boucrot et al., 2005) and © 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 639–647

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this interaction was shown to prevent the recruitment of kinesin to the SCV, thereby allowing the SCV to maintain a subcellular position permissive for Salmonella proliferation. Recent work by McGourty et al. (2012) reported the misrouting of immature Cathepsin to the secretory pathway by intracellular Salmonella. The SPI2-T3SSand SifA-dependent activity results in an impaired maturation of the SCV and protects intracellular Salmonella against antimicrobial enzymatic activities. The group observed the formation of a complex consisting of SifA, SKIP and Rab9. The sequestration of Rab9 to the SCV interferes with retrograde transport of vesicles containing immature lysosomal enzymes to the TGN. As a functional consequence, lysosomal enzymes are secreted, and detoxified lysosomes devoid of hydrolytic enzymes become available for fusion to the SCV (McGourty et al., 2012). Recently, PLEKHM1 was identified as a further host cell target for SifA (McEwan et al., 2015). Knockdown of PLEKHM1 resulted in reduced intracellular proliferation of Salmonella and the formation of large vacuoles containing many bacteria, in contrast to tight-fitting SCV with individual bacteria in control host cells. By deploying SifA, Salmonella recruits a complex of PLEKHM1, Rab7 and HOPS to SifA-containing membranes. This complex mediates the fusion of detoxified lysosomes to the SCV. SIF are highly dynamic compartments with a unique membrane organization. Commonly, light microscopy is used to analyse the presence or absence of canonical marker proteins of host cell compartments on the SCV. However, because of the diffraction limit of light microscopy, these approaches provide only limited insights into the spatial organization of the SCV and the biogenesis of SIT. Ultrastructural techniques, in particular transmission electron microscopy (TEM), provide sufficient spatial resolution to resolve the organization of SCV and SIT. A systematic ultrastructural analysis of Salmonella-infected cells revealed a number of novel and unexpected features of SCV and SIT that may revise previous models for the intracellular environment of Salmonella (Krieger et al., 2014). These findings are depicted in Fig. 1. The combined ultrastructural analysis involving TEM, EM tomography and live cell imaging correlated to EM (live CLEM) showed that SIF initiate as single-membrane tubular compartments (leading SIF) (Fig. 1H and I). During development, these tubules convert into double-membrane tubules (trailing SIF), whereas mutants lacking the SPI2 effector SseF or SseG fail to induce double-membrane SIF (Fig. 1J and K). While double-membrane vesicles are characteristic for macro-autophagy, double-membrane SIF lack canonical autophagy markers and their formation does not require a functional autophagy pathway.

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Intracellular Salmonella

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Fig. 1. Phenotypes induced by intracellular Salmonella enterica and interactions with host membrane compartments. A. Model of Salmonella intracellular lifestyle and interactions with host membrane compartments. After SPI1-T3SS-mediated invasion of host cells or phagocytic uptake, internalized Salmonella is located in a host-derived membrane compartment, termed SCV. A subpopulation of Salmonella escapes from the SCV and is targeted by macro-autophagy or occasionally hyper-replicates in the cytosol (C). By default, bacteria remain in an early SCV from which Salmonella-induced SVAT and SNX3 tubules extend. In parallel, the SCV moves along microtubules towards the MTOC. By means of the SPI2-T3SS, Salmonella translocates another set of effector proteins. This activity induces various types of SIT, i.e. SIF, SIST and LNT. SIT are formed through fusion processes with recruited vesicles derived from different host cell membrane compartments and move along microtubules. SIT membranes contain proteins from various cellular origins. Around the SCV vacuole-associated actin polymerizes. B. At later time points post infection (8–10 h post infection, p.i.) HeLa cells expressing LAMP1-GFP display a stable network of LAMP1-positive SIF, accompanied by a strong reduction of host cell endosomes. C. Inside some cells a subpopulation of Salmonella escapes from the SCV and hyper-replicates inside the cytosol. D–G. CLEM analyses of HeLa cells infected with Salmonella wild type (WT) 8 h p.i. revealed a double-membrane organization for LAMP1-positive SIF with cytosolic content inside the inner lumen (E) and Salmonella within the outer lumen (F, G). H and I. In the initial phase (4 h p.i.) of centrifugal extension, SIF consist of thinner single-membrane SIF at the tips (leading SIF) and thicker double-membrane SIF at the base (trailing SIF), as revealed by CLEM analyses. J and K. Cells infected with sseF or sseG mutant strains show thin SIF (J) with single-membrane organization (TEM micrographs, K). L–N. Pulse-chase experiments with infected HeLa cells show that the fluid phase CLEM marker bovine serum albumin (BSA)-Rhodamine is internalized into the host endo-lysosomal system and by means of Salmonella-induced rearrangement of vesicle traffic finally accumulates inside SCV (L, N) and SIF outer lumen (L, M). BSA-Rhodamine as CLEM marker can photo-convert diaminobenzidine (DAB) into an osmiophilic polymer visible on TEM sections. O. Salmonella WT-infected HeLa cells immunostained for LAMP1 and β-tubulin display microtubule bundling along SIF structures. P–Q. TEM micrographs of infected HeLa cells show microtubule-like structures (P) and F-actin-like structures (Q) inside SIF inner lumen. Abbreviations: Ap (autophagosome), EE (early endosome), ER (endoplasmic reticulum), LE (late endosome), LNT, LS (leading SIF), Lys (lysosome), Mc (mitochondria), Mt (microtubule), MTOC (microtubule-organizing centre), RE (recycling endosome), SCV, SIF, SIST, SIT, SNX3 tubule, SVAT, TGN, TS (trailing SIF), VAP (vacuole-associated actin polymerization). Scale bars: 10 μm; B, C, H, J, L, O; 2 μm, D; 500 nm, E, F, G, I, K, M, N, P, Q. Arrow heads: yellow, SIF outer membrane; orange, SIF inner membrane; dark blue, microtubules; light blue, F-actin. Figure parts were reproduced, with permission, from references Kuhle et al. (2004), Zhang and Hensel (2013) and Krieger et al. (2014).

However, double-membrane vesicles are also observed during replication of certain viruses (reviewed in Blanchard and Roingeard, 2015). The ultrastructural analyses identified the presence of ribosomes, small membrane vesicles and cytoskeletal filaments, i.e. microtubules and F-actin inside a subset of SIF (Fig. 1O and Q). These host cell structures were only observed in double-membrane SIF and their presence can be explained by a new model for SIF biogenesis (Krieger et al., 2014). SCV containing single or smaller clusters of bacteria were observed in connection with one or multiple SIF. Live CLEM also identified single Salmonella cells located in the space between the two SIF membranes, termed outer lumen, and these bacteria were frequently observed moving within the tubule (Fig. 1D–G). While light microscopy of fluid tracers did not provide sufficient spatial resolution to resolve the origin of the SIF inner and outer lumen, the ultrastructural analyses and cytochemistry clearly revealed that only the SIF outer lumen is in interchange with endocytosed fluid phase markers (Fig. 1L–N). New models for formation of SIF. Based on results of previous studies and novel observations for SIF ultrastructure, we propose a novel model for SIF biogenesis. The activity of SPI2-T3SS effector proteins, in particular SifA, results in continuous fusion of membrane vesicles to the SCV. SIF emerging from the SCV extend centrifugally along microtubules and activity of PipB2 as © 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 639–647

linker for kinesin promotes extension (Henry et al., 2006a; Rajashekar et al., 2014). Tubular extensions initiate as single-membrane SIF (leading SIF) and convert to double-membrane SIF (trailing SIF). This conversion requires the function of SseF and SseG (Krieger et al., 2014; Rajashekar et al., 2014). We propose that lateral extension of SIF membranes around the guiding microtubule filament results in entrapment of portions of cytosol, microtubules and/or F-actin. This process appears to be reversible in the initial phase, but over time, membrane closure occurs, resulting in completely closed double-membrane tubules. The inner lumen of this compartment is derived from cytoplasm as evidenced by the presence of cytoskeletal filaments, ribosomes and smaller vesicles. In contrast, the outer lumen is derived from the luminal content of vesicles that fused to SCV and SIF. The lumen of the SCV is connected to the lumen of singlemembrane SIF, as well as to the outer lumen of doublemembrane SIF. The connection of SCV lumen to the extensive network of SIF and other SIT may allow the continuous interchange of luminal content and will make luminal content available to Salmonella inside the SCV. Open questions regarding the intracellular environment of Salmonella The recent findings on heterogeneity and dynamics of SIT, their proteome composition and ultrastructural features give rise to various new hypotheses and open

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Fig. 2. Open questions regarding the intracellular lifestyle of Salmonella and the physiological role of SIT. A. What is the cellular origin of membranes integrated in SIT? Which Salmonella effectors are involved in recruitment of vesicles from different host cell compartments? B. Do vesicles recruited from the host cell fuse to the SCV, to tips of SIT or to SIT at any position? C. Is the intracellular activity of Salmonella resulting in the induction of additional tubular membrane compartments? D. How are double-membrane (DM) SIF formed? The SPI2-T3SS effector-mediated recruitment of host cells SNAREs (mediators of membrane fusions) and BAR proteins (mediators of membrane curvature) to SIT may result in membrane deformation and fusion. E. How is the entrapment of actin and microtubule filaments by DM SIF mediated and does the luminal cytoskeleton lead to a stabilization of DM SIF and decreased dynamics? F. How is the highly dynamic and partially reversible transition of leading SIF (LS) to trailing SIF (TS) (as shown in I) mediated? The lack of DM SIF in mutant strains defective in SseF or SseG indicates a specific contribution of these effector proteins. G. Does the SIT network provide a nutritional supply for Salmonella in the SCV? The connection of the lumen of the SCV to the SIF network could provide access to endocytosed compounds. H. Does the connection between the SCV and SIF network reduce the exposure to antimicrobial activities of the host cell. Accumulation of lytic enzymes and acidification of the SCV ultimately result in killing of intracellular bacteria. The connection of the SCV to the SIF network allows exchange of luminal content and dilution of antimicrobial activities. I. Dynamic properties of LS and TS in Salmonella-infected HeLa cells expressing LAMP1-GFP. Relative time-points are shown as h : min : seconds. Arrow heads: yellow, tip of TS; white, tip of LS. Scale bar: 1 μm. Figure reproduced, with permission, from Rajashekar et al. (2008).

questions to be solved by future experimental analyses (see Fig. 2 for visual details and further explanation). What is the cellular origin and composition of membrane vesicles recruited to the SCV? For example, recent work indicated that lgp-positive membrane lacking the lysosomal enzyme activities are recruited to the SCV, rather than functional lysosomes (McGourty et al., 2012). This question may be addressed by following the interac-

tion of distinct host cell organelles with the SCV with high temporal and spatial resolution. Where does vesicular fusion to SCV and/or SIF take place? Pulse/chase experiments with endocytic tracers may be used to follow the fate of endosomes in Salmonella-infected cells but have experimental limitations. As the relatively small volume of endosomal compartments merges with the large volume of SCV and © 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 639–647

Intracellular Salmonella SIF lumen, fusion events usually result in a rapid loss of fluorescence signal. Do further types of SIT exist in addition to SIF, SIST, LNT, SVAT and SNX3 tubules described so far? The various intracellular pathogens have evolved specialized lifestyles, but Salmonella is unique in inducing the extensive tubular networks. Use of further organelles markers for live cell analyses of host cells may reveal additional membrane compartments with altered morphology upon Salmonella infection. Is the extension of SIF tubules guided by cytoskeleton and is the cytoskeleton later enclosed by the membrane tubules? Such entrapment of cytoskeleton may result in stabilization of SIF and decrease in dynamics observed at later stage of infection. This may also explain the previous finding of microtubule bundling (Kuhle et al., 2004). Multiple filaments may become entrapped in the inner lumen of double-membrane SIF. The role of microtubule motor proteins for SIF formation has been previously observed (Henry et al., 2006b). Future work may investigate if interference with motor protein functions also results in absence of cytoskeleton within double-membrane SIF. Which host cell mechanisms mediate the membrane fusion during double-membrane formation? Membrane curvature and vesicle fusions are highly regulated processes in mammalian cells and controlled by BAR (Bin– Amphiphysin–Rvs) domain and SNARE (Soluble NSF Attachment Protein Receptor) proteins respectively. The conversion of single-membrane to double-membrane tubules may indicate a continuous zipper-like closure mechanism. It is open to speculation how the conversion of single- to double-membrane tubules is mediated, and which host cell factors are involved in this process. Such factors could be identified by further proteome analyses or functional interference studies, such as siRNA-mediated knock-down of defined components of the host cell vesicle fusion machinery. How does the composition in the SCV lumen change if SIT extension initiates? The microenvironment of Salmonella in the SCV has been characterized by various previous studies and acidification of the SCV lumen was observed (Martin-Orozco et al., 2006). However, most of the analyses were performed on ensembles rather than on the single-cell level. The recent observations of the interconnected SIF network give raise to the question if an acidic pH is maintained in the SCV. The continuous fusion with various types of vesicles may result in the mixing of luminal content, resulting in neutralization of pH. This mechanism may also affect other antimicrobial factors acting on Salmonella within the SCV. One may speculate that the increased volume of the interconnected SIT network results in the quenching of an initially acidic pH, as well as in the dilution of luminal content with antimicro© 2015 John Wiley & Sons Ltd, Cellular Microbiology, 17, 639–647

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bial activities. The pH of the SCV needs to be determined during various stages of formation of the SIT network and in absence of SIT. Are SIT required for nutrition of Salmonella within the SCV? Mutant strains defective in the SPI2-T3SS are highly attenuated in virulence in animal models. This attenuation is reflected by highly reduced intracellular replication in cell culture models for intracellular pathogenesis. The virulence attenuation in vivo and in vitro may, in part, be the result of an impaired defence of intracellular Salmonella against antimicrobial activities. In addition, we propose here a more basal explanation for the attenuation of SPI2-T3SS mutant strains: The lack of SIT induction secludes intracellular Salmonella from access to nutrients. The highly reduced intracellular replication thus may simply reflect nutritional limitation and inability in biosynthesis of molecules required for cell growth and division. The interconnected nature of the SIT network and the SCV may allow free diffusion of endosomal cargo within this network and may make this content accessible to Salmonella within the SCV. Future work should reveal if induction of the SIF network supports intracellular nutrition of Salmonella by accessing endosomal cargo. Conclusions and outlook Salmonella enterica has adapted to life inside mammalian host cells in a highly evolved manner. Many basic host cell functions are manipulated for the sake of the pathogen’s intracellular survival and replication. Understanding these virulence mechanisms will unravel the intricacies of the host–pathogen interaction and may open new avenues for prevention and treatment of systemic infections. The multifaceted nature and dynamics of Salmonella–host cell interaction call for live cell analyses of infection. An important complication of the study of the cellular microbiology of Salmonella is the heterogeneity in the activities of individual intracellular bacteria (reviewed in Helaine and Holden, 2013). This heterogeneity necessitates analyses of the activities of intracellular Salmonella on a single-cell level and the systematic spatial and temporal tracking of individual bacteria to analyse their intracellular fate. Does the cellular microbiology of intracellular Salmonella reflect the pathogenesis of systemic Salmonella infections in mammalian hosts? Most studies were done in cancer cells such as HeLa or immortalized cell lines and induction of SIT has been observed in various cellular infection models. There is also strong correlation between SPI2-T3SS function in intracellular replication and SIT formation in cellular infection models and systemic pathogenesis in animal models. Very limited information about the situation in vivo is available and experimental

646 V. Liss and M. Hensel evidence for the remodelling of the endosomal system in tissue cells is pending. This may be due to the limitation in spatial resolution of imaging and the detrimental effects of chemical fixation on tubular membrane compartments. A recently established neonate murine infection model (Zhang et al., 2014) may provide an interesting alternative to previously used animal models. Confirmation of the cellular phenotypes by analyses of Salmonella-infected tissue cells is needed, but such analyses are expected to be technically extremely demanding. Combined efforts to analyse the dynamic properties of Salmonella by live cell microscopy, the features of its intracellular compartments by ultrastructural analyses, and the composition of SCV/SIT compartments by proteomics/lipidomics should lead to new insights into the specific intracellular lifestyle of Salmonella.

Acknowledgements We apologize to all authors whose works were not discussed here because of space limitations. Work in our group was supported by grants HE1964/17-1, HE1964/18-2 within the priority programme SPP 1580 of the DFG and SFB 944.

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