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Growth control in the Salmonella-containing vacuole
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Growth control in the Salmonella-containing vacuole Francisco Garcı´a-del Portillo, Cristina Nu´n˜ez-Herna´ndez, Blanca Eisman and Jose´ Ramos-Vivas Salmonella enterica is an intracellular bacterial pathogen that inhabits membrane-bound vacuoles of eukaryotic cells. Coined as the ‘Salmonella-containing vacuole’ (SCV), this compartment has been studied for two decades as a replicative niche. Recent findings reveal, however, marked differences in the lifestyle of bacteria enclosed in the SCV of varied host cell types. In fibroblasts, the emerging view supports a model of bacteria facing in the SCV a ‘to grow’ or ‘not to grow’ dilemma, which is solved by entering in a dormancy-like state. Finetuning of host cell defense/survival routes, drastic metabolic shift down, adaptation to hypoxia conditions, and attenuation of own virulence systems emerge as strategies used by Salmonella to intentionally reduce the growth rate inside the SCV. Addresses Departamento de Biotecnologı´a Microbiana, Centro Nacional de Biotecnologı´a, Consejo Superior de Investigaciones Cientı´ficas (CSIC), Darwin 3, 28049 Madrid, Spain Corresponding author: Garcı´a-del Portillo, Francisco ([email protected])
Current Opinion in Microbiology 2008, 11:46–52 This review comes from a themed issue on Bacteria Edited by Thomas Meyer and Ilan Rosenshine Available online 20th February 2008 1369-5274/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2008.01.001
Introduction Pathogens replicate and propagate in susceptible hosts. In most cases, extensive replication is, however, not required to ensure dissemination. In fact, many successful pathogens displaying high infection rates prevent overt damage and coexist with the host for long-lasting (even life-long) periods. Bacterial pathogens with such ability include Mycobacterium tuberculosis, Helicobacter pylori, and Salmonella enterica [1–3]. Host defenses, intricate strategies developed by the pathogen, or a combination of both, ultimately impact the capacity of the pathogen to proliferate in host tissues. How pathogens attenuate their replication rate in host tissues is still poorly understood, though some mechanistic details start to emerge. For example, hypoxia and nitric oxide (NO) production by the host predispose M. tuberculosis for entering a nonreCurrent Opinion in Microbiology 2008, 11:46–52
plicative dormant state in lung granulomas. Two bacterial kinase proteins, DevS and DevT, act as hypoxia and redox sensors to activate a cognate response regulator, DevR, which ultimately reprograms gene expression to initiate dormancy [4,5]. S. enterica has necessarily evolved mechanisms to restrain growth in the host. This pathogen causes gastroenteritis and systemic diseases in humans and animals but is often associated with asymptomatic infections. At the cellular level, S. enterica lives within membrane-bound vacuoles and causes acute and long-lasting infections by surviving and replicating preferentially in macrophages [6,7]. Other cells targeted during the infection are epithelial, dendritic, and fibroblast-like cells. To establish a chronic infection, different genes are used by the pathogen at defined stages [8]. In this complex scenario, temporal and spatial modeling of the Salmonella-containing vacuole (SCV) is expected to occur. This review summarizes recent findings revealing marked differences in the SCV of diverse host cell types. Unlike the evidence found in other nonphagocytic cell types, S. enterica produces a long-lasting infection inside the SCV of fibroblasts with very limited, if any, proliferation (Figure 1) [9]. Recent transcriptome assays have shed light on how this unique lifestyle may have evolved. Interestingly, some of the unveiled new traits resemble those of the M. tuberculosis dormancy state [10].
Salmonella, vacuoles, cytosol, and autophagy The SCV has been intensively studied in tissue culture models [11–13]. Except in dendritic cells and fibroblasts, intravacuolar bacteria have been shown to replicate inside the SCV. Intriguingly, S. enterica does not undergo many replication rounds within the cells of animal tissues, neither in acute nor in chronic infections (reviewed in [12]). This picture implies the probable existence of mechanisms restricting replication, but not survival, of bacteria located inside the SCV. After 24 hours of infection, increases of no higher than twofold to threefold in intracellular wild-type bacteria are usually observed in fibroblasts [12]. However, bacterial mutants defective in certain functions, like the virulence regulatory system PhoP–PhoQ, overgrow in the SCV (Figure 1). Fibroblasts therefore exemplify a niche in which S. enterica probably adapts to an environment not supporting active growth. The view of the SCV as a compartment supporting only limited bacterial growth is not exclusive of fibroblasts. A www.sciencedirect.com
Salmonella growth inside vacuoles Garcı´a-del Portillo et al. 47
Figure 1
Distinct modes of Salmonella growth inside different nonphagocytic cell types. Shown is a comparison of the Salmonella lifestyle inside epithelial cells and fibroblasts. Intracellular bacteria initiate the infection enclosed in the Salmonella-containing vacuole (SCV). In epithelial cells, this compartment is further modeled (1) to sustain bacterial proliferation (2). Salmonella replicates, however, at higher rates in the cytosol of epithelial cells, showing a characteristic larger cell size as it has been shown for wild-type or sifA strains (3). In epithelial cells, no differences have been reported in the phenotypes of wild-type and phoP bacteria. In fibroblasts, wild-type bacteria adapt to a nonproliferative state inside the SCV (4). By contrast, mutants with an inactive PhoP–PhoQ system overgrow inside the SCV upon the infection of this particular cell type (5). A proportion of SCV (damaged) and cytosolic bacteria is proposed to be targeted by the autophagic system and ubiquitinated proteins in both cell types (6). The significance of these later processes for the control of Salmonella intracellular growth remains to be determined. Relevant to the control of Salmonella intracellular growth is the action of host (TBK-1) and bacterial (SifA) proteins that ensure integrity of the SCV membrane. Very recently, the AKT-1 (Akt) kinase has also been implicated in preventing fusion of the SCV with lysosomes of epithelial cells, promoting bacterial replication in this way. This later model, however, contrasts with the elevated rate of interaction with the endolysosomal system shown for the SCV in HeLa epithelial cells.
defect in SifA, a bacterial protein secreted by the type-III secretion system encoded in the pathogenicity island-2 (SPI-2 TTSS), results in release of many intravacuolar bacteria to the cytosol [14,15]. Lack of SifA has consequences for growth since bacteria replicate at higher rates in the cytosol than inside the SCV of epithelial cells. Wild-type bacteria (0.7–24%, depending on the study) have also been claimed to reside in the cytosol upon release from damaged SCV [14,15]. The in vivo significance of this vacuole-to-cytosol transition and the subsequent massive cytosolic replication remain, however, elusive. Thus, though S. enterica replicates efficiently in animal tissues during an acute infection, such process occurs essentially by net increase in infection foci rather than by undergoing massive replication within cells [16]. www.sciencedirect.com
Recently, it has been shown that TANK-binding kinase 1 (TBK-1), a host factor involved in type-I interferon responses mediating antiviral-defense, preserves the integrity of the SCV [17]. tbk1( / ) embryonic fibroblasts contain more intracellular bacteria, predominantly located in the cytosol. This increase in bacterial load occurs in different cell types in which TBK-1 was knocked out (epithelial, fibroblasts, and macrophages) as well as in tbk1( / ) fibroblasts infected with other pathogens as Streptococcus pyogenes and pathogenic Escherichia coli [17]. These results are in line with the data obtained with the sifA mutant and favor the hypothesis that the SCV environment, in spite of supporting bacterial growth in certain cell types, could be more restrictive than the host cell cytosol. Recent work has shown that in epithelial cells the autophagosomal marker LC3 (light chain 3) colocalizes with intracellular S. enterica, and that such event peaks at onehour postinfection [18]. Most bacteria having LC3-labeling retain colocalization with SCV-membrane proteins as Lamp-1, which suggests that damaged (perforated) SCVs are rapidly targeted by the autophagic system [19]. Perforation of the SCV membrane could be accomplished by the invasion-associated type-III secretion apparatus encoded in the pathogenicity island-1 (SPI-1 TSS) [20], which remains active in intracellular bacteria. However, only 20% of the SCV is surrounded by LC3 in infected epithelial cells displaying maximal autophagic activity [19]. So, it remains to be demonstrated whether bacteria enclosed in the SCV of epithelial cells subvert autophagy or not. In macrophages, S. enterica induces autophagy and cell death by injecting SipB, an effector protein secreted by SPI-1 TTSS [21]. Autophagy, as a process responding to stress conditions [22], modulates the survival of M. tuberculosis inside macrophages [23,24]. At steady-state, about 5% of M. tuberculosis-containing vacuoles colocalize with LC3 whereas 35% of these vacuoles become positive upon autophagy stimulation by either nutritional starvation or drugs [22]. Importantly, autophagy releases the maturation arrest imposed by M. tuberculosis on its vacuole [23]. Intravacuolar survival of M. tuberculosis is also controlled by interferon-g (INF-g). Thus, immunity-related GTPases (IRGs) activated upon INF-g stimulation promote antimycobacterial effect by inducing autophagy [24,25]. Remarkably, no study has yet addressed whether autophagy induction alters the trafficking route of the SCV and the growth or survival of intravacuolar S. enterica. This aspect could be addressed in freshly isolated macrophages, which impair bacterial survival inside the SCV. Interestingly, S. enterica proliferates at a large extent in atg5( / ) embryonic fibroblasts, deficient in autophagosome maturation [19]. These differences correlate with the presence of a larger population of cytosolic bacteria, which were otherwise decorated with ubiquitinated proteins as in Current Opinion in Microbiology 2008, 11:46–52
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wild-type fibroblasts. M. tuberculosis survival is affected by the exposure to ubiquitin-derived peptides [26], but it is unknown whether a similar mechanism applies for S. enterica in fibroblasts. Studies in macrophages have shown that cytosolic wild-type bacteria colocalize with ubiquitinated proteins, a process that may ultimately contribute to pathogen death [27]. The exact mechanism of this proposed ubiquitin-mediated killing has not been explored further. Furthermore, increased cytosolic positioning and growth of S. enterica in the autophagy-defective atg5( / ) fibroblasts is intriguing considering that cytosol extracts of fibroblasts restrict S. enterica growth [14]. Finally, it should be noted that pathogens as Shigella flexneri and Listeria monocytogenes, which reside in the cytosol, efficiently evade autophagy [28–30]. In summary, in the case of S. enterica infections these yet-confusing data do not clarify whether autophagy limits bacterial growth in the SCV and how the process is directed.
Alarming but preserving the host cell Besides autophagy, eukaryotic cells use other tools to combat bacterial infections, like the pattern-recognition receptors (PRRs) [31] or members of the mitogen-activated protein (MAP) kinase family [32]. MAP kinase cascades are activated by mitogenic stimuli such as growth factors and hormones (ERK 1/2 pathway); or by a variety of cellular and environmental stresses (JNK and p38 pathways). In macrophages, S. enterica and M. tuberculosis activate extracellular signal-regulated kinases 1/2 (ERK 1/2), which impairs pathogen intravacuolar growth [33,34]. S. enterica also activates ERK 1/2 kinases in fibroblasts (Eisman et al., unpublished). Consistent to these observations, mutants overgrowing in the SCV of fibroblasts activate the ERK 1/2 cascade to a lesser extent. Intravacuolar wild-type bacteria may therefore intentionally stimulate these MAP kinases to attenuate the growth rate. It is noteworthy that neither the JNK nor the p38 cascades are activated in S. enterica-infected fibroblasts (Eisman et al., unpublished), contrasting to the activation of these MAP kinases reported in epithelial cells infected with S. enterica or Campylobacter jejuni [35,36]. These differences reinforce once more the varied lifestyle of S. enterica in the SCV of distinct host cell types. In epithelial cells, S. enterica activates the pro-survival antiapoptotic kinase Akt-1 (also known as protein kinase B, PKB) by injecting SopB/SigD, a secreted effector with phosphoinositide-phosphatase activity. Although the exact mechanism of activation remains undefined, Akt1 is activated by S. enterica in a sustained manner several hours after bacterial entry [37]. This mode of activation is proposed to ensure the survival of the infected cell and to facilitate subsequent bacterial proliferation within the SCV. Such strategy has implications for the intestinal infection since S. enterica encounters epithelial cells undergoing a rapid turnover and having Akt-1 normally downregulated. S. enterica may also activate Akt-1 to limit Current Opinion in Microbiology 2008, 11:46–52
inflammation and ensure intestinal colonization [38]. In fibroblasts, nongrowing wild-type bacteria activate Akt-1 to a greater extent than mutants that overgrow inside the SCV (Eisman et al., unpublished). Apoptosis inhibition could facilitate long-lasting persistence of the bacteria in this particular SCV. Pharmacological inhibition of ERK 1/ 2-phosphorylation or Akt-1-phosphorylation results in overgrowth of wild-type S. enterica inside the SCV of fibroblasts, which is consistent with active host defenses operating in this nonphagocytic cell type. Taken together, these observations further support the idea of S. enterica intentionally stimulating host defenses to attenuate growth in the SCV.
Nutrition inside the SCV The microenvironment of the SCV remains largely unknown. However, recent studies have shed light on nutrients and intermediate metabolism probably used by S. enterica inside the SCV. Microarray analysis performed with serovar Typhimurium in murine macrophages indicated that magnesium, manganese, and iron could be limited in the SCV compared to a nutrient-rich medium like LB (reviewed in [39]). Genes encoding transporters required for the uptake of these cations were upregulated in intracellular bacteria. Using a proteomic approach, Shi et al. confirmed that some of these transporters, such as MgtB, SitA, SitB, and IroN, are abundant in bacteria recovered from macrophages [40]. Intriguingly, a recent microarray study suggests that iron may be not limiting in the SCV containing serovar Typhi [41]. This particular SCV may not have limiting manganese or phosphate, inferred by the non-induction of the sitABCD operon or genes induced by phosphate deprivation as phoN, pstA, or pstB [41]. Strikingly, serovar Typhi, but not Typhimurium, upregulates aceA in the SCV of macrophages. aceA encodes isocitrate lyase, an enzyme involved in the metabolism of fatty acids and acetate as carbon sources via the glyoxylate shunt [42]. These differences infer distinct lifestyles for these two serovars in the SCV of macrophages. Variables as the source of macrophages, infection conditions, methods of RNA isolation, and comparators used (bacteria growing in LB or collected from infection growth medium), may also affect the relative extent of these differences. As an example, the microarray analysis performed with serovar Typhimurium used macrophages expressing a nonfunctional Slc11a1 (also known as Nramp or Ity) protein. Slc11a1 is a metal efflux pump proposed to remove essential cations from the SCV and therefore it is relevant in the control of bacterial intracellular growth [43]. In addition, these studies were performed with actively growing bacteria, which may differ in physiological terms from bacteria nonreplicating or struggling for survival in the SCV. Such comparison imposes a technical challenge because of the limitations of obtaining sufficient material from nongrowing populations of intracellular bacteria. www.sciencedirect.com
Salmonella growth inside vacuoles Garcı´a-del Portillo et al. 49
Regarding to in vivo data, recent proteomic studies identified serovar Typhimurium proteins in tissue extracts of infected mice and inferred usage of both aerobic and anaerobic electron acceptors [44]. Through a systematic analysis of bacterial mutants lacking key metabolic enzymes, Tchawa Yimga et al. showed that sugars could be used as main carbon source and that the tricarboxylic acid (TCA) cycle may operate as full cycle in the acute infection [45]. Fang et al. recently demonstrated that, for establishing a chronic infection, serovar Typhimurium may use fatty acids and acetate as carbon sources via the glyoxylate shunt [46]. AceA was shown to be essential in this infection model. Nonreplicating serovar Typhimurium also upregulates the glyoxylate shunt in cultured fibroblasts. Likewise, M. tuberculosis has two isocitrate lyases (Icl1 and Icl2) and both are involved in persistence and virulence [47]. Regarding other metabolic parameters, M. tuberculosis may undergo different respiratory states depending on the infection stage [48]. During the chronic infection, nitrate is apparently preferred as terminal electron acceptor [10]. The nonreplicative phase of S. enterica in fibroblasts is also accompanied by the upregulation of reductases using alternative electron acceptors other than oxygen (Nu´n˜ez-Herna´ndez et al., unpublished). Nonreplicating serovar Typhimurium inhabiting the SCV of fibroblasts display other metabolic traits resembling those reported for ‘dormant’ M. tuberculosis [10] (Figure 2). These include the decreased expression of genes encoding components of the H+-ATPase pump (atp operon) and ribosomal proteins as well as the upregulation of enzymes involved in anaerobic lipid catabolism and gluconeogenesis. Another important environmental cue unveiled in the SCV of fibroblasts is hypoxia (Nu´n˜ez-Herna´ndez et al., unpublished) [9]. This condition triggers entry of M. tuberculosis in the dormant state [4,5]. Cyo and Cyd terminal oxidases, which use oxygen exclusively as terminal acceptor, are strongly downregulated in bacteria inhabiting the SCV of fibroblasts. S. enterica also reduces in this niche energy-dependent processes as flagella synthesis, consistent with the probable reduction of protonmotive force (PMF) and the aforementioned probable downregulation of the H+-ATPase pump. This lowenergy state would also explain the strong upregulation of the ‘phage-shock-response’ ( psp) genes, which are induced upon PMF decrease [49]. Intriguingly, in fibroblasts S. enterica also upregulates genes that are under control of the alternative sigma factor RpoN (sN or s54), including some genes encoding enhancer-binding proteins (EBPs) promoting transcription by the RpoN–RNA–polymerase complex [50]. Target genes of the sN–EBP regulon are mostly involved in responses allowing survival in hostile environments, including adaptation to niches with limiting nitrogen sources. psp genes form part of the sN–EBP regulon www.sciencedirect.com
Figure 2
Main physiological changes deciphered in nonreplicating Salmonella enterica enclosed in the SCV of fibroblasts. (a) Components of S. enterica growing extracellularly that are strongly downregulated upon entry into fibroblasts; (b) functions upregulated in bacteria remaining in a nonreplicative state inside the SCV. Some environmental cues assigned to this particular SCV, as hypoxia and ions limitation, are also indicated. In blue are marked changes also described for dormant Mycobacterium tuberculosis (hypoxia; usage of lipids acetate as carbon source via the glyoxylate shunt; and upregulation of alternative reductases). Abbreviations: AceA, isocitrate lyase; EBP, enhancer-binding proteins that mediate RpoN (sN)-dependent transcription; Psp, phage-shock proteins; SPI-1–SPI-2 TTSS, type-III secretion systems encoded in the pathogenicity islands SPI-1 and SPI-2.
and are also highly upregulated by S. enterica inhabiting the SCV of macrophages [39,41] and fibroblasts.
Expression of virulence genes by nongrowing S. enterica The specialized SPI-2 TTSS secretion system is a key virulence factor largely involved in intracellular replication and survival within macrophage and epithelial cells [51,52]. In fibroblasts, expression of SPI-2 genes has been Current Opinion in Microbiology 2008, 11:46–52
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observed in nongrowing (wild-type) and overgrowing bacteria ( phoP mutant) (Nu´n˜ez-Herna´ndez et al., unpublished). Unlike what has been reported in macrophages [53], bacteria residing within the fibroblast may not strictly require a functional PhoP–PhoQ system to induce the SPI-2 TTSS system. Expression of SPI-2 genes also occurs in nongrowing Salmonella located inside the SCV of dendritic cells [54], though it is unknown the relative contribution of the PhoP–PhoQ in this particular cell type. The fact that in fibroblasts the exacerbated growth of the phoP mutant relies on a functional SPI-2 TTSS [55] sustains a model based on the fine-tuning of this virulence system as strategy to modulate the growth rate inside the SCV.
could prevent fusion of the SCV with lysosomes. Thus, pharmacological inhibition of Akt-1 as well as other kinases in a same functional network was shown to impair replication of Salmonella within the SCV. Akt-1 was previously shown to be targeted by Salmonella to promote antiapoptotic routes in the infected epithelial cell [37]. It is noteworthy that recent work by Drecktrah et al. in HeLa epithelial cells [58] revealed that the SCV, though being a replicative niche in this cell type, interacts with late compartments of the endocytic route such as lysosomes. Therefore, the exact extent at which Salmonella inhibits SCV–lysosome fusion in epithelial cells and its impact on the control of bacterial growth remains to be clarified.
Conclusions
Acknowledgements
S. enterica establishes different ways of life inside distinct eukaryotic cell types. Until recently, the biology of SCVs harboring nonreplicating bacteria was largely ignored. However, recent studies have shed new lights on this particular condition, finely regulated by the pathogen. The data collected in the fibroblast model reveal a remarkable overlap with data previously reported for M. tuberculosis in the dormant, nonreplicative state. Environmental cues and responses shared in both conditions include hypoxia, downregulation of the ATPsynthase, usage of fatty acids as main carbon source, and the synthesis of proteins responding to stress (Figure 2). A yet missing element in the S. enterica model is a dedicated ‘regulatory system’ promoting the adaptation to a nonreplicative state, as the DevSTR system in M. tuberculosis. If existing, the identification of such novel regulatory network would be undoubtedly a major breakthrough to understanding how S. enterica controls intravacuolar growth. Finally, we should be more aware of the often-discordant data obtained in distinct in vitro cultured cells and in vivo mouse models, which probably reflects the inherent limitations of both systems. Future studies directly addressing the ultimate basis of the divergent and common trends found are clearly required. These analyses would increase our limited knowledge of the processes behind the disparate growth rates observed in different cell types and infection conditions.
We thank Olivia Steele-Mortimer for the critical comments on sections of this manuscript. Work in the laboratory of FGP is supported by grants BIO2004-03455-C02-01 and GEN2006-27776-C2-1-E/PAT from the Ministry of Education and Science of Spain and LSHB-CT-2005-512061 (NoE ‘EuroPathogenomics’) from the European Union. CNH is recipient of a Fellowship from the ‘Consejerı´a de Educacio´n de la Comunidad de Madrid’.
Update Very recently, two studies have provided new insights on the different lifestyle of Salmonella inside the SCV of host distinct cell types as well as in mechanisms that could control the extent of bacterial replication within the SCV. In a microarray analysis performed in HeLa epithelial cells, Hautefort et al. [56] uncovered that, unlike what was previously observed in macrophages, Salmonella growing inside the SCV expresses the three type-III secretion systems present in this pathogen (flagella and those encoded in SPI-1 and SPI-2). In the second study, Kuijl et al. [57] report using breast cancer epithelial cell that the targeting by SopB of PKB (Akt-1) Current Opinion in Microbiology 2008, 11:46–52
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macrophages revealed through the selective capture of transcribed sequences. Proc Natl Acad Sci U S A 2006, 103:1906-1911. 42. Munoz-Elias EJ, McKinney JD: Carbon metabolism of intracellular bacteria. Cell Microbiol 2006, 8:10-22. Excellent review that describes the current knowledge on the metabolism of bacteria inhabiting the intracellular niche of eukaryotic cells or animal locations such as the intestine. It highlights the profound differences found with metabolism known for laboratory-grown bacteria. 43. Fortier A, Min-Oo G, Forbes J, Lam-Yuk-Tseung S, Gros P: Single gene effects in mouse models of host:pathogen interactions. J Leukoc Biol 2005, 77:868-877. 44. Becker D, Selbach M, Rollenhagen C, Ballmaier M, Meyer TF, Mann M, Bumann D: Robust Salmonella metabolism limits possibilities for new antimicrobials. Nature 2006, 440:303-307. Systematic identification by proteomics of Salmonella enterica proteins synthesized during acute infection of susceptible mice, making an analysis of the preferred metabolic routes used in vivo. 45. Tchawa Yimga M, Leatham MP, Allen JH, Laux DC, Conway T, Cohen PS: Role of gluconeogenesis and the tricarboxylic acid cycle in the virulence of Salmonella enterica serovar Typhimurium in BALB/c mice. Infect Immun 2006, 74:11301140. 46. Fang FC, Libby SJ, Castor ME, Fung AM: Isocitrate lyase (AceA) is required for Salmonella persistence but not for acute lethal infection in mice. Infect Immun 2005, 73:2547-2549. This study shows that isocitrate lyase (AceA) is an important factor in the Salmonella chronic infection. 47. Munoz-Elias EJ, McKinney JD: Mycobacterium tuberculosis isocitrate lyases 1 and 2 are jointly required for in vivo growth and virulence. Nat Med 2005, 11:638-644. 48. Shi L, Sohaskey CD, Kana BD, Dawes S, North RJ, Mizrahi V, Gennaro ML: Changes in energy metabolism of Mycobacterium tuberculosis in mouse lung and under in vitro conditions affecting aerobic respiration. Proc Natl Acad Sci U S A 2005, 102:15629-15634. 49. Darwin AJ: The phage-shock-protein response. Mol Microbiol 2005, 57:621-628. 50. Studholme DJ: Enhancer-dependent transcription in Salmonella enterica Typhimurium: new members of the sigmaN regulon inferred from protein sequence homology and predicted promoter sites. J Mol Microbiol Biotechnol 2002, 4:367-374.
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51. Abrahams GL, Hensel M: Manipulating cellular transport and immune responses: dynamic interactions between intracellular Salmonella enterica and its host cells. Cell Microbiol 2006, 8:728-737. 52. Waterman SR, Holden DW: Functions and effectors of the Salmonella pathogenicity island 2 type III secretion system. Cell Microbiol 2003, 5:501-511. 53. Bijlsma JJ, Groisman EA: The PhoP/PhoQ system controls the intramacrophage type three secretion system of Salmonella enterica. Mol Microbiol 2005, 57:85-96. 54. Jantsch J, Cheminay C, Chakravortty D, Lindig T, Hein J, Hensel M: Intracellular activities of Salmonella enterica in murine dendritic cells. Cell Microbiol 2003, 5:933-945. 55. Cano DA, Martinez-Moya M, Pucciarelli MG, Groisman EA, Casadesus J, Garcia-Del Portillo F: Salmonella enterica serovar Typhimurium response involved in attenuation of pathogen intracellular proliferation. Infect Immun 2001, 69:6463-6474. 56. Hautefort I, Thompson A, Eriksson-Ygberg S, Parker ML, Lucchini S, Danino V, Bongaerts RJ, Ahmad N, Rhen M, Hinton JC: During infection of epithelial cells Salmonella enterica serovar Typhimurium undergoes a time-dependent transcriptional adaptation that results in simultaneous expression of three type 3 secretion systems. Cell Microbiol 2008, 10:958-984. By analysis of genome expression of Salmonella upon infection of Hela epithelial cells, authors show common and unique features compared to intracellular bacteria in the SCV of macrophages. A striking finding was the simultaneous expression of three type-III secretion systems in intracellular bacteria (flagella and those encoded by SPI-1 and SPI-2). 57. Kuijl C, Savage ND, Marsman M, Tuin AW, Janssen L, Egan DA, Ketema M, van den Nieuwendijk R, van den Eeden SJ, Geluk A et al.: Intracellular bacterial growth is controlled by a kinase network around PKB/AKT1. Nature 2007, 450:725-730. This study analyses the effect on Salmonella replication of distinct drugs in epithelial cells that target kinases and proposes that AKT-1 (PKB) plays a pivotal role in the control of intracellular growth by preventing fusion of the SCV with lysosomes. 58. Drecktrah D, Knodler LA, Howe D, Steele-Mortimer O: Salmonella trafficking is defined by continuous dynamic interactions with the endolysosomal system. Traffic 2007, 8:212-225. This work demonstrates in HeLa epithelial cells that the SCV, modeled as replicative niche in this cell type, is a highly dynamic compartment that interacts with late compartments of the endocytic route such as lysosomes, even at early infection times.
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Growth control in the Salmonella-containing vacuole Francisco Garcı´a-del Portillo, Cristina Nu´n˜ez-Herna´ndez, Blanca Eisman and Jose´ Ramos-Vivas Salmonella enterica is an intracellular bacterial pathogen that inhabits membrane-bound vacuoles of eukaryotic cells. Coined as the ‘Salmonella-containing vacuole’ (SCV), this compartment has been studied for two decades as a replicative niche. Recent findings reveal, however, marked differences in the lifestyle of bacteria enclosed in the SCV of varied host cell types. In fibroblasts, the emerging view supports a model of bacteria facing in the SCV a ‘to grow’ or ‘not to grow’ dilemma, which is solved by entering in a dormancy-like state. Finetuning of host cell defense/survival routes, drastic metabolic shift down, adaptation to hypoxia conditions, and attenuation of own virulence systems emerge as strategies used by Salmonella to intentionally reduce the growth rate inside the SCV. Addresses Departamento de Biotecnologı´a Microbiana, Centro Nacional de Biotecnologı´a, Consejo Superior de Investigaciones Cientı´ficas (CSIC), Darwin 3, 28049 Madrid, Spain Corresponding author: Garcı´a-del Portillo, Francisco ([email protected])
Current Opinion in Microbiology 2008, 11:46–52 This review comes from a themed issue on Bacteria Edited by Thomas Meyer and Ilan Rosenshine Available online 20th February 2008 1369-5274/$ – see front matter # 2008 Elsevier Ltd. All rights reserved. DOI 10.1016/j.mib.2008.01.001
Introduction Pathogens replicate and propagate in susceptible hosts. In most cases, extensive replication is, however, not required to ensure dissemination. In fact, many successful pathogens displaying high infection rates prevent overt damage and coexist with the host for long-lasting (even life-long) periods. Bacterial pathogens with such ability include Mycobacterium tuberculosis, Helicobacter pylori, and Salmonella enterica [1–3]. Host defenses, intricate strategies developed by the pathogen, or a combination of both, ultimately impact the capacity of the pathogen to proliferate in host tissues. How pathogens attenuate their replication rate in host tissues is still poorly understood, though some mechanistic details start to emerge. For example, hypoxia and nitric oxide (NO) production by the host predispose M. tuberculosis for entering a nonreCurrent Opinion in Microbiology 2008, 11:46–52
plicative dormant state in lung granulomas. Two bacterial kinase proteins, DevS and DevT, act as hypoxia and redox sensors to activate a cognate response regulator, DevR, which ultimately reprograms gene expression to initiate dormancy [4,5]. S. enterica has necessarily evolved mechanisms to restrain growth in the host. This pathogen causes gastroenteritis and systemic diseases in humans and animals but is often associated with asymptomatic infections. At the cellular level, S. enterica lives within membrane-bound vacuoles and causes acute and long-lasting infections by surviving and replicating preferentially in macrophages [6,7]. Other cells targeted during the infection are epithelial, dendritic, and fibroblast-like cells. To establish a chronic infection, different genes are used by the pathogen at defined stages [8]. In this complex scenario, temporal and spatial modeling of the Salmonella-containing vacuole (SCV) is expected to occur. This review summarizes recent findings revealing marked differences in the SCV of diverse host cell types. Unlike the evidence found in other nonphagocytic cell types, S. enterica produces a long-lasting infection inside the SCV of fibroblasts with very limited, if any, proliferation (Figure 1) [9]. Recent transcriptome assays have shed light on how this unique lifestyle may have evolved. Interestingly, some of the unveiled new traits resemble those of the M. tuberculosis dormancy state [10].
Salmonella, vacuoles, cytosol, and autophagy The SCV has been intensively studied in tissue culture models [11–13]. Except in dendritic cells and fibroblasts, intravacuolar bacteria have been shown to replicate inside the SCV. Intriguingly, S. enterica does not undergo many replication rounds within the cells of animal tissues, neither in acute nor in chronic infections (reviewed in [12]). This picture implies the probable existence of mechanisms restricting replication, but not survival, of bacteria located inside the SCV. After 24 hours of infection, increases of no higher than twofold to threefold in intracellular wild-type bacteria are usually observed in fibroblasts [12]. However, bacterial mutants defective in certain functions, like the virulence regulatory system PhoP–PhoQ, overgrow in the SCV (Figure 1). Fibroblasts therefore exemplify a niche in which S. enterica probably adapts to an environment not supporting active growth. The view of the SCV as a compartment supporting only limited bacterial growth is not exclusive of fibroblasts. A www.sciencedirect.com
Salmonella growth inside vacuoles Garcı´a-del Portillo et al. 47
Figure 1
Distinct modes of Salmonella growth inside different nonphagocytic cell types. Shown is a comparison of the Salmonella lifestyle inside epithelial cells and fibroblasts. Intracellular bacteria initiate the infection enclosed in the Salmonella-containing vacuole (SCV). In epithelial cells, this compartment is further modeled (1) to sustain bacterial proliferation (2). Salmonella replicates, however, at higher rates in the cytosol of epithelial cells, showing a characteristic larger cell size as it has been shown for wild-type or sifA strains (3). In epithelial cells, no differences have been reported in the phenotypes of wild-type and phoP bacteria. In fibroblasts, wild-type bacteria adapt to a nonproliferative state inside the SCV (4). By contrast, mutants with an inactive PhoP–PhoQ system overgrow inside the SCV upon the infection of this particular cell type (5). A proportion of SCV (damaged) and cytosolic bacteria is proposed to be targeted by the autophagic system and ubiquitinated proteins in both cell types (6). The significance of these later processes for the control of Salmonella intracellular growth remains to be determined. Relevant to the control of Salmonella intracellular growth is the action of host (TBK-1) and bacterial (SifA) proteins that ensure integrity of the SCV membrane. Very recently, the AKT-1 (Akt) kinase has also been implicated in preventing fusion of the SCV with lysosomes of epithelial cells, promoting bacterial replication in this way. This later model, however, contrasts with the elevated rate of interaction with the endolysosomal system shown for the SCV in HeLa epithelial cells.
defect in SifA, a bacterial protein secreted by the type-III secretion system encoded in the pathogenicity island-2 (SPI-2 TTSS), results in release of many intravacuolar bacteria to the cytosol [14,15]. Lack of SifA has consequences for growth since bacteria replicate at higher rates in the cytosol than inside the SCV of epithelial cells. Wild-type bacteria (0.7–24%, depending on the study) have also been claimed to reside in the cytosol upon release from damaged SCV [14,15]. The in vivo significance of this vacuole-to-cytosol transition and the subsequent massive cytosolic replication remain, however, elusive. Thus, though S. enterica replicates efficiently in animal tissues during an acute infection, such process occurs essentially by net increase in infection foci rather than by undergoing massive replication within cells [16]. www.sciencedirect.com
Recently, it has been shown that TANK-binding kinase 1 (TBK-1), a host factor involved in type-I interferon responses mediating antiviral-defense, preserves the integrity of the SCV [17]. tbk1( / ) embryonic fibroblasts contain more intracellular bacteria, predominantly located in the cytosol. This increase in bacterial load occurs in different cell types in which TBK-1 was knocked out (epithelial, fibroblasts, and macrophages) as well as in tbk1( / ) fibroblasts infected with other pathogens as Streptococcus pyogenes and pathogenic Escherichia coli [17]. These results are in line with the data obtained with the sifA mutant and favor the hypothesis that the SCV environment, in spite of supporting bacterial growth in certain cell types, could be more restrictive than the host cell cytosol. Recent work has shown that in epithelial cells the autophagosomal marker LC3 (light chain 3) colocalizes with intracellular S. enterica, and that such event peaks at onehour postinfection [18]. Most bacteria having LC3-labeling retain colocalization with SCV-membrane proteins as Lamp-1, which suggests that damaged (perforated) SCVs are rapidly targeted by the autophagic system [19]. Perforation of the SCV membrane could be accomplished by the invasion-associated type-III secretion apparatus encoded in the pathogenicity island-1 (SPI-1 TSS) [20], which remains active in intracellular bacteria. However, only 20% of the SCV is surrounded by LC3 in infected epithelial cells displaying maximal autophagic activity [19]. So, it remains to be demonstrated whether bacteria enclosed in the SCV of epithelial cells subvert autophagy or not. In macrophages, S. enterica induces autophagy and cell death by injecting SipB, an effector protein secreted by SPI-1 TTSS [21]. Autophagy, as a process responding to stress conditions [22], modulates the survival of M. tuberculosis inside macrophages [23,24]. At steady-state, about 5% of M. tuberculosis-containing vacuoles colocalize with LC3 whereas 35% of these vacuoles become positive upon autophagy stimulation by either nutritional starvation or drugs [22]. Importantly, autophagy releases the maturation arrest imposed by M. tuberculosis on its vacuole [23]. Intravacuolar survival of M. tuberculosis is also controlled by interferon-g (INF-g). Thus, immunity-related GTPases (IRGs) activated upon INF-g stimulation promote antimycobacterial effect by inducing autophagy [24,25]. Remarkably, no study has yet addressed whether autophagy induction alters the trafficking route of the SCV and the growth or survival of intravacuolar S. enterica. This aspect could be addressed in freshly isolated macrophages, which impair bacterial survival inside the SCV. Interestingly, S. enterica proliferates at a large extent in atg5( / ) embryonic fibroblasts, deficient in autophagosome maturation [19]. These differences correlate with the presence of a larger population of cytosolic bacteria, which were otherwise decorated with ubiquitinated proteins as in Current Opinion in Microbiology 2008, 11:46–52
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wild-type fibroblasts. M. tuberculosis survival is affected by the exposure to ubiquitin-derived peptides [26], but it is unknown whether a similar mechanism applies for S. enterica in fibroblasts. Studies in macrophages have shown that cytosolic wild-type bacteria colocalize with ubiquitinated proteins, a process that may ultimately contribute to pathogen death [27]. The exact mechanism of this proposed ubiquitin-mediated killing has not been explored further. Furthermore, increased cytosolic positioning and growth of S. enterica in the autophagy-defective atg5( / ) fibroblasts is intriguing considering that cytosol extracts of fibroblasts restrict S. enterica growth [14]. Finally, it should be noted that pathogens as Shigella flexneri and Listeria monocytogenes, which reside in the cytosol, efficiently evade autophagy [28–30]. In summary, in the case of S. enterica infections these yet-confusing data do not clarify whether autophagy limits bacterial growth in the SCV and how the process is directed.
Alarming but preserving the host cell Besides autophagy, eukaryotic cells use other tools to combat bacterial infections, like the pattern-recognition receptors (PRRs) [31] or members of the mitogen-activated protein (MAP) kinase family [32]. MAP kinase cascades are activated by mitogenic stimuli such as growth factors and hormones (ERK 1/2 pathway); or by a variety of cellular and environmental stresses (JNK and p38 pathways). In macrophages, S. enterica and M. tuberculosis activate extracellular signal-regulated kinases 1/2 (ERK 1/2), which impairs pathogen intravacuolar growth [33,34]. S. enterica also activates ERK 1/2 kinases in fibroblasts (Eisman et al., unpublished). Consistent to these observations, mutants overgrowing in the SCV of fibroblasts activate the ERK 1/2 cascade to a lesser extent. Intravacuolar wild-type bacteria may therefore intentionally stimulate these MAP kinases to attenuate the growth rate. It is noteworthy that neither the JNK nor the p38 cascades are activated in S. enterica-infected fibroblasts (Eisman et al., unpublished), contrasting to the activation of these MAP kinases reported in epithelial cells infected with S. enterica or Campylobacter jejuni [35,36]. These differences reinforce once more the varied lifestyle of S. enterica in the SCV of distinct host cell types. In epithelial cells, S. enterica activates the pro-survival antiapoptotic kinase Akt-1 (also known as protein kinase B, PKB) by injecting SopB/SigD, a secreted effector with phosphoinositide-phosphatase activity. Although the exact mechanism of activation remains undefined, Akt1 is activated by S. enterica in a sustained manner several hours after bacterial entry [37]. This mode of activation is proposed to ensure the survival of the infected cell and to facilitate subsequent bacterial proliferation within the SCV. Such strategy has implications for the intestinal infection since S. enterica encounters epithelial cells undergoing a rapid turnover and having Akt-1 normally downregulated. S. enterica may also activate Akt-1 to limit Current Opinion in Microbiology 2008, 11:46–52
inflammation and ensure intestinal colonization [38]. In fibroblasts, nongrowing wild-type bacteria activate Akt-1 to a greater extent than mutants that overgrow inside the SCV (Eisman et al., unpublished). Apoptosis inhibition could facilitate long-lasting persistence of the bacteria in this particular SCV. Pharmacological inhibition of ERK 1/ 2-phosphorylation or Akt-1-phosphorylation results in overgrowth of wild-type S. enterica inside the SCV of fibroblasts, which is consistent with active host defenses operating in this nonphagocytic cell type. Taken together, these observations further support the idea of S. enterica intentionally stimulating host defenses to attenuate growth in the SCV.
Nutrition inside the SCV The microenvironment of the SCV remains largely unknown. However, recent studies have shed light on nutrients and intermediate metabolism probably used by S. enterica inside the SCV. Microarray analysis performed with serovar Typhimurium in murine macrophages indicated that magnesium, manganese, and iron could be limited in the SCV compared to a nutrient-rich medium like LB (reviewed in [39]). Genes encoding transporters required for the uptake of these cations were upregulated in intracellular bacteria. Using a proteomic approach, Shi et al. confirmed that some of these transporters, such as MgtB, SitA, SitB, and IroN, are abundant in bacteria recovered from macrophages [40]. Intriguingly, a recent microarray study suggests that iron may be not limiting in the SCV containing serovar Typhi [41]. This particular SCV may not have limiting manganese or phosphate, inferred by the non-induction of the sitABCD operon or genes induced by phosphate deprivation as phoN, pstA, or pstB [41]. Strikingly, serovar Typhi, but not Typhimurium, upregulates aceA in the SCV of macrophages. aceA encodes isocitrate lyase, an enzyme involved in the metabolism of fatty acids and acetate as carbon sources via the glyoxylate shunt [42]. These differences infer distinct lifestyles for these two serovars in the SCV of macrophages. Variables as the source of macrophages, infection conditions, methods of RNA isolation, and comparators used (bacteria growing in LB or collected from infection growth medium), may also affect the relative extent of these differences. As an example, the microarray analysis performed with serovar Typhimurium used macrophages expressing a nonfunctional Slc11a1 (also known as Nramp or Ity) protein. Slc11a1 is a metal efflux pump proposed to remove essential cations from the SCV and therefore it is relevant in the control of bacterial intracellular growth [43]. In addition, these studies were performed with actively growing bacteria, which may differ in physiological terms from bacteria nonreplicating or struggling for survival in the SCV. Such comparison imposes a technical challenge because of the limitations of obtaining sufficient material from nongrowing populations of intracellular bacteria. www.sciencedirect.com
Salmonella growth inside vacuoles Garcı´a-del Portillo et al. 49
Regarding to in vivo data, recent proteomic studies identified serovar Typhimurium proteins in tissue extracts of infected mice and inferred usage of both aerobic and anaerobic electron acceptors [44]. Through a systematic analysis of bacterial mutants lacking key metabolic enzymes, Tchawa Yimga et al. showed that sugars could be used as main carbon source and that the tricarboxylic acid (TCA) cycle may operate as full cycle in the acute infection [45]. Fang et al. recently demonstrated that, for establishing a chronic infection, serovar Typhimurium may use fatty acids and acetate as carbon sources via the glyoxylate shunt [46]. AceA was shown to be essential in this infection model. Nonreplicating serovar Typhimurium also upregulates the glyoxylate shunt in cultured fibroblasts. Likewise, M. tuberculosis has two isocitrate lyases (Icl1 and Icl2) and both are involved in persistence and virulence [47]. Regarding other metabolic parameters, M. tuberculosis may undergo different respiratory states depending on the infection stage [48]. During the chronic infection, nitrate is apparently preferred as terminal electron acceptor [10]. The nonreplicative phase of S. enterica in fibroblasts is also accompanied by the upregulation of reductases using alternative electron acceptors other than oxygen (Nu´n˜ez-Herna´ndez et al., unpublished). Nonreplicating serovar Typhimurium inhabiting the SCV of fibroblasts display other metabolic traits resembling those reported for ‘dormant’ M. tuberculosis [10] (Figure 2). These include the decreased expression of genes encoding components of the H+-ATPase pump (atp operon) and ribosomal proteins as well as the upregulation of enzymes involved in anaerobic lipid catabolism and gluconeogenesis. Another important environmental cue unveiled in the SCV of fibroblasts is hypoxia (Nu´n˜ez-Herna´ndez et al., unpublished) [9]. This condition triggers entry of M. tuberculosis in the dormant state [4,5]. Cyo and Cyd terminal oxidases, which use oxygen exclusively as terminal acceptor, are strongly downregulated in bacteria inhabiting the SCV of fibroblasts. S. enterica also reduces in this niche energy-dependent processes as flagella synthesis, consistent with the probable reduction of protonmotive force (PMF) and the aforementioned probable downregulation of the H+-ATPase pump. This lowenergy state would also explain the strong upregulation of the ‘phage-shock-response’ ( psp) genes, which are induced upon PMF decrease [49]. Intriguingly, in fibroblasts S. enterica also upregulates genes that are under control of the alternative sigma factor RpoN (sN or s54), including some genes encoding enhancer-binding proteins (EBPs) promoting transcription by the RpoN–RNA–polymerase complex [50]. Target genes of the sN–EBP regulon are mostly involved in responses allowing survival in hostile environments, including adaptation to niches with limiting nitrogen sources. psp genes form part of the sN–EBP regulon www.sciencedirect.com
Figure 2
Main physiological changes deciphered in nonreplicating Salmonella enterica enclosed in the SCV of fibroblasts. (a) Components of S. enterica growing extracellularly that are strongly downregulated upon entry into fibroblasts; (b) functions upregulated in bacteria remaining in a nonreplicative state inside the SCV. Some environmental cues assigned to this particular SCV, as hypoxia and ions limitation, are also indicated. In blue are marked changes also described for dormant Mycobacterium tuberculosis (hypoxia; usage of lipids acetate as carbon source via the glyoxylate shunt; and upregulation of alternative reductases). Abbreviations: AceA, isocitrate lyase; EBP, enhancer-binding proteins that mediate RpoN (sN)-dependent transcription; Psp, phage-shock proteins; SPI-1–SPI-2 TTSS, type-III secretion systems encoded in the pathogenicity islands SPI-1 and SPI-2.
and are also highly upregulated by S. enterica inhabiting the SCV of macrophages [39,41] and fibroblasts.
Expression of virulence genes by nongrowing S. enterica The specialized SPI-2 TTSS secretion system is a key virulence factor largely involved in intracellular replication and survival within macrophage and epithelial cells [51,52]. In fibroblasts, expression of SPI-2 genes has been Current Opinion in Microbiology 2008, 11:46–52
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observed in nongrowing (wild-type) and overgrowing bacteria ( phoP mutant) (Nu´n˜ez-Herna´ndez et al., unpublished). Unlike what has been reported in macrophages [53], bacteria residing within the fibroblast may not strictly require a functional PhoP–PhoQ system to induce the SPI-2 TTSS system. Expression of SPI-2 genes also occurs in nongrowing Salmonella located inside the SCV of dendritic cells [54], though it is unknown the relative contribution of the PhoP–PhoQ in this particular cell type. The fact that in fibroblasts the exacerbated growth of the phoP mutant relies on a functional SPI-2 TTSS [55] sustains a model based on the fine-tuning of this virulence system as strategy to modulate the growth rate inside the SCV.
could prevent fusion of the SCV with lysosomes. Thus, pharmacological inhibition of Akt-1 as well as other kinases in a same functional network was shown to impair replication of Salmonella within the SCV. Akt-1 was previously shown to be targeted by Salmonella to promote antiapoptotic routes in the infected epithelial cell [37]. It is noteworthy that recent work by Drecktrah et al. in HeLa epithelial cells [58] revealed that the SCV, though being a replicative niche in this cell type, interacts with late compartments of the endocytic route such as lysosomes. Therefore, the exact extent at which Salmonella inhibits SCV–lysosome fusion in epithelial cells and its impact on the control of bacterial growth remains to be clarified.
Conclusions
Acknowledgements
S. enterica establishes different ways of life inside distinct eukaryotic cell types. Until recently, the biology of SCVs harboring nonreplicating bacteria was largely ignored. However, recent studies have shed new lights on this particular condition, finely regulated by the pathogen. The data collected in the fibroblast model reveal a remarkable overlap with data previously reported for M. tuberculosis in the dormant, nonreplicative state. Environmental cues and responses shared in both conditions include hypoxia, downregulation of the ATPsynthase, usage of fatty acids as main carbon source, and the synthesis of proteins responding to stress (Figure 2). A yet missing element in the S. enterica model is a dedicated ‘regulatory system’ promoting the adaptation to a nonreplicative state, as the DevSTR system in M. tuberculosis. If existing, the identification of such novel regulatory network would be undoubtedly a major breakthrough to understanding how S. enterica controls intravacuolar growth. Finally, we should be more aware of the often-discordant data obtained in distinct in vitro cultured cells and in vivo mouse models, which probably reflects the inherent limitations of both systems. Future studies directly addressing the ultimate basis of the divergent and common trends found are clearly required. These analyses would increase our limited knowledge of the processes behind the disparate growth rates observed in different cell types and infection conditions.
We thank Olivia Steele-Mortimer for the critical comments on sections of this manuscript. Work in the laboratory of FGP is supported by grants BIO2004-03455-C02-01 and GEN2006-27776-C2-1-E/PAT from the Ministry of Education and Science of Spain and LSHB-CT-2005-512061 (NoE ‘EuroPathogenomics’) from the European Union. CNH is recipient of a Fellowship from the ‘Consejerı´a de Educacio´n de la Comunidad de Madrid’.
Update Very recently, two studies have provided new insights on the different lifestyle of Salmonella inside the SCV of host distinct cell types as well as in mechanisms that could control the extent of bacterial replication within the SCV. In a microarray analysis performed in HeLa epithelial cells, Hautefort et al. [56] uncovered that, unlike what was previously observed in macrophages, Salmonella growing inside the SCV expresses the three type-III secretion systems present in this pathogen (flagella and those encoded in SPI-1 and SPI-2). In the second study, Kuijl et al. [57] report using breast cancer epithelial cell that the targeting by SopB of PKB (Akt-1) Current Opinion in Microbiology 2008, 11:46–52
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