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Caspase-1 activation by Salmonella Harri A. Ja¨rvela¨inen, Antoine Galmiche and Arturo Zychlinsky Max Planck Institute for Infection Biology, Department of Cellular Microbiology, Campus Charite´ Mitte, Schumannstraße 21/22, Berlin 10117, Germany.
Salmonella is an interesting example of how the selective pressure of host environments has led to the evolution of sophisticated bacterial virulence mechanisms. This microbe exploits the first-line of defence, the macrophage, as a crucial tool in the initiation of disease. After invasion of intestinal macrophages, a virulence protein secreted by Salmonella specifically induces apoptotic cell death by activating the cysteine protease caspase-1. The pro-apoptotic capability is necessary for successful pathogenesis. The study of mechanisms by which Salmonella induces programmed cell death offers new insights into how pathogens cause disease and into general mechanisms of activation of the innate immune system. Apoptosis is a genetically programmed cell event, in which the dying cell participates actively in its own death. It is evolutionarily conserved and found in all multicellular organisms, where it is essential for normal development and homeostasis. Apoptotic cell death results from activation of a cascade of enzymes called caspases [1]. Caspases are cysteine proteases, each having a characteristic role in the apoptotic process. Upstream initiator caspases are synthesized as inactive zymogens that usually are activated through regulated protein – protein interactions. The downstream effector caspases are cleaved by the initiator caspase in order to generate a catalytically active enzyme. Apoptosis of host cells is observed in many different bacterial infections [2]. Depending on the bacteria, host cell apoptosis can be detrimental or advantageous to pathogenesis. Apoptosis often plays an important role in defence against intracellular pathogens, preventing the use of a safe niche within the host. For example, bronchial cell apoptosis mediated by the cell death receptor CD95 leads to increased survival of the host during murine Pseudomonas aeruginosa pneumonia, demonstrating the in vivo relevance of apoptosis in host defence against P. aeruginosa [3]. Salmonella, however, initiates apoptosis as a virulence strategy. Salmonellosis Salmonellae are Gram-negative facultative intracellular bacteria. Different Salmonella species cause a wide range of symptoms, ranging from a mild intestinal infection to life-threatening systemic infections. After ingestion, virulent Salmonella use principally M cells, specialized antigen-sampling epithelial cells lining the lymphoid Corresponding author: Arturo Zychlinsky ([email protected]).
follicles of the intestines, to cross the intestinal barrier (Fig. 1) [4,5]. Once inside the tissue, Salmonella infects macrophages, activates apoptosis and provokes mucosal inflammation. Bacteria are then transported via the lymphatics to mesenteric lymph nodes, which serve as a ‘distribution centre’ before Salmonella disseminates through the blood. Then, during several subsequent days, the bacteria reside and replicate intracellularly in phagocytic cells of the spleen and liver [6]. Salmonella pathogenesis can be studied with S. typhimurium infections in mice. Mice infected orally reproduce many, but not all, of the symptoms seen in the course of typhoid fever in humans [7,8]. Virulence mechanisms and intracellular life During cell invasion, Salmonella hijacks both the cytoskeleton and the membrane trafficking machinery of its target cells. At the site of cell contact, Salmonella effector proteins induce macropinocytic activities that change the membrane profoundly. The actin cytoskeleton rearrangements, which lead to Salmonella internalization, are achieved by the activation of host GTPases of the Rho family [9]. Salmonella survives and replicates within a unique vacuolar compartment called the ‘Salmonellacontaining vacuole’ derived from the late endosomal compartment of the cell [10,11]. Approximately 4% of the Salmonella genome, around 180 genes, is related to virulence. Many virulence genes are encoded in two large clusters within the Salmonella genome, Salmonella Pathogenicity Island (SPI) 1 and SPI2 [12 – 14]. These pathogenicity islands encode the components of two type III secretion apparatus, specialized secretion systems allowing the delivery of bacterial effector proteins inside the host cell [15]. Upon cell contact, this complex machinery is thought to form needle-like structures spanning the inner and outer membranes of the bacterial cell. Mutations in either one of these virulence islands lead to an attenuated infection in mice, but in different fashions (Fig. 1). Virulence genes in SPI1 are primarily expressed during the initial gastrointestinal phase of the infection. The SPI1 genes allow bacteria to infiltrate and establish infection within the epithelium and intestinal lymphoid tissues [16]. SPI1 is not necessary for the systemic phase of the infection as SPI1 mutants are not attenuated when they are administered intraperitoneally in mice, a model that bypasses the gastrointestinal phase. By contrast, genes encoded in SPI2 are necessary in the systemic phase of the infection as they enable the bacteria to survive and replicate within macrophages and other host cells [14,17].
http://ticb.trends.com 0962-8924/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0962-8924(03)00032-1
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Fig. 1. The natural history of a Salmonella infection. Genes encoded by the Salmonella Pathogenicity Island-1 and -2 (SPI1 and SPI2) have different roles during the course of Salmonella infection. The functions of SPI1 are necessary primarily during the intestinal phase of the infection (phase I), ranging from bacterial adherence and invasion to intracellular life in intestinal lymphoid tissues. The genes of SPI2 are crucial in the systemic phase of the disease (phase II), during which bacteria remain within macrophages in the spleen and liver.
Mutants in SPI2 are able to infect Peyer’s patches but fail to progress to the mesenteric lymph nodes. The early gastrointestinal phase of the infection is accompanied by the induction of acute mucosal inflammation [18]. This inflammation, as we will discuss later, requires the interaction of SPI1-encoded virulence protein Salmonella invasion protein B (SipB) with host cysteine protease caspase-1 [19]. SipB is required for the translocation of proteins through the type III secretion apparatus. Whether SipB is a ‘protein translocase’ in the membrane of host cells is still controversial [9,20]. SipB, however, plays an important intracellular role during the infection of eukaryotic cells. SipB is homologous (41% amino acid identity) and functionally analogous to the virulence factor IpaB produced by Shigella spp., a related organism that causes dysentery [21]. Although IpaB is thought to form pores in the plasma membrane of eukaryotic cells [22], neither pore formation nor hemolytic activity have been described for Salmonella. SipB is organized into two functional domains that probably account for the membrane-interaction properties. The amino-terminus might promote self-multimerization, whereas the carboxy-terminus interacts with membrane lipids [23]. Indeed, SipB appears to partition to membranes through its carboxy-terminal domain [23]. A topological analysis of SipB in phosphatidylcholine-containing liposomes suggests that SipB could span a complete bilayer [24]. It is still unclear whether this phenomenon is relevant for protein function in vivo. A comparative structure–function analysis of highly homologous regions of IpaB suggests that this might be the case [25]. A study linking the in vitro hydrophobic interactions of SipB and their role during Salmonella infections is warranted. Caspases Caspase-1 belongs to a mammalian family of caspases that consists of at least 14 members (Fig. 2) [26]. Each caspase http://ticb.trends.com
has a characteristic role in the initiation or execution of cell disassembly. Accordingly, they have been grouped into families known as ‘initiators’ and ‘executioners’. A diverse set of signals from outside and inside the cell can activate initiator caspases (including caspases-8, -9 and -10). Caspase-8 or -10-dependent pathways are triggered by their recruitment to the transmembrane domain of cellsurface death receptors (such as the TNF type 1 receptor family). Various forms of cellular stress, inducing p2
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Fig. 2. Phylogenetic classification of selected mammalian caspases (adapted from [39]). Effector caspases (such as caspase-3) contain no prodomain, whereas other members have either a death effector domain (DED, purple) or a CARD (caspase recruitment domain, yellow). CARD-containing caspases include caspase-1, caspase-5 (human) and caspase-11 (mouse). The catalytic domain of caspases shares significant structural homology among the various caspases: it contains a large 20-kDa and a small 10-kDa unit. Both of these subunits contribute four surface loops (L1– L4, blue) that shape the catalytic groove.
Functions of caspase-1 Caspase-1-dependent programmed cell death is distinct from other forms of ‘classical apoptosis’ as measured by at least three characteristics. First, the key elements of the mitochondrial pathway, such as caspase-3 and the proteins Bcl-2 and Bcl-xL, are not required in this cell death pathway [28]. Components of the caspase-1 apoptotic pathway are not known. One candidate could be raf-1, which is degraded during Salmonellainduced cell death, but the phenotype of raf-1-null macrophages is weak in Salmonella infections [29]. As caspase-3 is not involved in the caspase-1-dependent pathway, cleavage of poly (ADP-ribose) polymerase-1 (PARP-1) does not take place [18]. Second, in contrast to knockout mice of other caspases, which present with several developmental problems, caspase-1 knockout mice develop normally [30]. However, cells deficient in caspase-1 undergo apoptosis in response to many of the ‘classical’ apoptotic stimuli, such as UV irradiation or treatment with staurosporine [31]. Third, paradoxically, although apoptosis is thought to be an ‘immunologically silent’ process, caspase-1 is a proinflammatory enzyme. Caspase-1 cleaves the inactive precursors of IL-1b and IL-18, generating their mature active forms [32,33]. These cytokines do not contain the signal sequences of secretory proteins. It is still unclear how they are released from the cell after cleavage by caspase-1. Secretion of IL-1b might involve a nonclassical secretory pathway with exocytosis of lysosomal-related organelles [34] or plasma membrane vesiculation [35]. Alternatively, apoptosis itself might be required. Regulation of caspase-1 activation Protein – protein interactions most likely play a central part in the regulation of caspase-1. Martinon et al. [36] recently described that, in macrophages, caspase-1 is activated in a complex called the inflammasome. This large complex (more than 700 kDa) contains caspase-1 and caspase-5 as well as the adaptor proteins NALP1 and Pycard. These adaptors are essential in the assembly of the inflammasome. They contain either a Caspase Recruitment Domain (CARD) or the related Pyrin domain (PYD). These domains are found in the long prodomains of initiator caspases and belong to the death-domain-fold http://ticb.trends.com
superfamily, which includes the death domain (DD) and the death effector domain (DED) family [37]. Despite sharing little sequence identity, they fold into similar three-dimensional structures comprising six anti-parallel alpha helices. Both the CARD and PYD domains are able, like the DD and DED, to form homotypic interactions. The CARD or PYD domains interact highly specifically only with other CARD or PYD domains. In the case of the inflammasome, the CARD and PYD domains support the assembly of the complex by bringing together its protein components, including caspase-1 and caspase-5. The inflammasome is reminiscent of another caspase-activating complex of high molecular mass (1.4 MDa) – the apoptosome. The apoptosome drives the activation of caspase-9 following the release of cytochrome c from the mitochondrial intermembrane space. The resolution of the three-dimensional structure of the apoptosome revealed that it is a wheelshaped oligomer [38]. In addition to caspase-9, this complex contains the adaptor protein Apaf-1 (apoptotic protease-activating factor). In the presence of ATP or dATP, Apaf-1 binds to cytochrome c, triggering Apaf-1 selfmultimerization and formation of a platform on which caspase-9 docks and is activated [39]. Similarly to Apaf-1, NALP-1 contains a nucleotidebinding domain (NBD) (Fig. 3). This domain defines a family of factors, NODs (nucleotide-binding oligomerization
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cytochrome c release from the mitochondria, can initiate a ‘mitochondrial pathway’ leading to caspase-9 activation. Executioner caspases (such as caspase-3 and -7) are usually activated by proteolytic cleavage between domains by a previously activated upstream caspase. All caspases have three domains: an amino-terminal prodomain, and a catalytic domain, which consists of two subunits with shared structural homology among caspases (a large 20-kDa and a small 10-kDa unit). Cleavage usually occurs between the prodomain and the p20 domain after Asp residues. Therefore, the processing is believed to occur mostly autocatalytically. The affinity of each caspase for different substrates can vary considerably [27]. Ultimately, cell death takes place owing to the proteolytic cleavage of various cellular targets by activated caspases.
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Fig. 3. Regulatory proteins of caspase-1. Proteins listed have been documented to either activate or downregulate caspase-1 activity. The precursor of caspase-1 is activated by a proteolytic cleavage in the linker region between the amino-terminal CARD domain (yellow) and the two catalytic subunits (p20 and p10). Catalytic residues (including the cysteine at position 285) are represented here in purple. The CARD and pyrin (see text) domains are shown in green. These proteins use their pyrin or CARD domains to modulate the assembly of a caspase-1-activating platform, recently named the inflammasome (see text). A subset of adaptor proteins containing nucleotide-binding oligomerization domains (NODs), NALP1/CARD7, Ipaf/CARD12/Clan and Pypaf7, contain a centrally located nucleotide-binding site (NBS) in addition to repeated leucine-rich repeat sequences at their C-termini. Because these repeated sequences are homologous to those presumed to recognize bacterial structures in the Toll-like receptors (TLRs), it has been proposed that NOD proteins might function as cytoplasmic sensors for bacterial compounds.
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domains) that play a crucial role in caspase activation. NODs share a common structural organization, with an amino-terminal CARD/PYD domain, a centrally located NBD and repeated sequences at their carboxy-termini [40]. Interestingly, the leucine-rich repeats in NALP-1 are similar to those found in Toll-like receptors (TLRs), which recognize molecules common to many microbes [41]. Indeed, NODs appear to function as cytoplasmic sensors for microbial compounds [42,43]. Different microbial molecules and endogenous proinflammatory stimuli activate macrophages to release IL-1b. To what extent these proinflammatory stimuli induce apoptosis is not well characterized. An interesting case is the activation of TLRs, which under certain conditions can signal for apoptosis, and the release of IL-1b. In this case, apoptosis is dependent on caspase-8, but not caspase-1, whereas the release of mature IL-1b is dependent on caspase-1 [44]. Salmonella-induced cell death Salmonella-induced cell death requires caspase-1 activity as it is prevented by specific inhibitors (ValAla-Asp-fluoromethylketone; YVAD-FMK) and is not observed in macrophages derived from caspase-1knockout mice. A direct role for SipB in apoptosis is supported by the observation that microinjection of recombinant SipB protein is sufficient to induce apoptosis in macrophages [19]. However, the mechanism of caspase-1 activation by SipB is not clear. Binding studies performed with GST– SipB or immunoprecipitation experiments suggest that SipB binds to caspase-1 [19]. A direct contact could promote a change in caspase-1 conformation. Alternatively, SipB could help establish a local concentration of caspase-1 by favouring its auto-activation (a ‘nucleation’ effect) [39]. Interestingly, at high multiplicity of infection, cell death can be observed in caspase-1-deficient macrophages. Whether this represents a physiologically relevant pathway remains to be determined. It is possible that, in the absence of caspase-1, SipB can activate a closely related caspase [45]. Salmonella also induces SPI2-dependent, SPI1-independent apoptosis of macrophages [46,47]. This apoptosis is partly dependent on caspase-1 [47], but the bacterial effector protein has not yet been identified. In comparison with SipB-mediated apoptosis, the execution of SPI2-dependent cell death is considerably delayed (18 – 24 h post infection). Morphologically, Salmonella-induced cell death is characterized by classical apoptotic changes – formation of large cytoplasmic vacuoles, membrane blebs, chromatin condensation and cell shrinkage (Fig. 4) [18]. One parameter that has been used to characterize host cell death is the delay between the loss of phosphatidylserine (PtdSer) asymmetry in the plasma membrane and the subsequent destruction of membrane integrity [48]. Early apoptotic cells display PtdSer in their outer leaflet while maintaining membrane integrity. Monack et al. elegantly showed the kinetics of these steps using time-lapse video microscopy of Salmonella-infected macrophages: during infection of macrophages with Salmonella, binding of annexin-V to the PtdSer in the outer leaflet of the http://ticb.trends.com
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Fig. 4. Salmonella typhimurium-infected macrophages show classical apoptotic morphology. (a) Electron-microscopic features of apoptosis. Arrows show membrane blebbing and arrowheads show Salmonella-containing vacuoles. Chromatin condensation and margination can be seen in the nucleus. (b) The DNA-binding dye propidium iodide (red) allows the visualization of chromatin and intracellular bacteria. Following the induction of apoptosis, chromatin condensation occurs (globular shape in the nucleus). The dying cell exposes phosphatidylserine (PtdSer), an aminophospholipid normally restricted to the inner leaflet of the plasma membrane, on the cell surface. Annexin V (green) specifically binds to PtdSer [48].
plasma membrane precedes the ultimate loss of macrophage membrane integrity, although the interval between these two parameters is very short (approximately 2 minutes; see http://cmgm.stanford.edu/falkow/index.html). The fact that Salmonella-induced cell death exhibits unique features (see ‘functions of caspase-1’) not seen in ‘classical’ apoptosis has led some authors to compare it to another form of cell death, necrosis [49 – 51]. The aspects of distinctive features of Salmonella-induced cell death and their resemblance to necrosis have been recently discussed in detail [18]. The dogma that apoptosis is a single entity has recently been challenged. Clearly, there are many alternative pathways for the execution of programmed cell death, leading to varying apoptotic phenotypes [52].
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Caspase-1-mediated apoptosis in Salmonella infection in vivo The most convincing evidence for the crucial role of caspase-1 in salmonellosis comes from studies using knockout mice. A1000-fold higher dose of Salmonella is required to lethally infect caspase-1-knockout mice compared with wild-type mice [53]. In caspase-1-knockout mice, Salmonella infects the Peyer’s patch only transiently and it does not spread to mesenteric lymph nodes and spleen. However, when the mice are infected intraperitoneally, a route that bypasses the intestine, the caspase-1null mice do not present a phenotype [53]. Because these phenotypes are reminiscent of the one obtained after infection with SPI1 or sipB mutants [54], SipB has been suggested to be the main activator for caspase-1 during in vivo infection. However, it is difficult to directly address this possibility because of the multiple roles of SipB in addition to directly activating caspase-1. Although these in vivo studies clearly indicate that caspase-1 is required for the systemic spread of Salmonella from the Peyer’s patches, the functions of apoptosis are not clear. The most direct interpretation of the data is that, since macrophages can kill bacteria, in caspase-1null mice, the bacteria, rather than the macrophages, are killed. However, this might not be the case because the intracellular survival of Salmonella does not differ between wild-type and caspase-1-null macrophages in vitro [53]. It is possible that IL-1b and IL-18, the two cytokines activated by caspase-1, are necessary for the intestinal inflammation characteristic of salmonellosis. The inflammatory response might compromise the integrity of the intestinal barrier, resulting in a secondary bacterial invasion from the intestines. Indeed, in a mouse lung model of Shigella infection, the profound phenotype observed in infections of caspase-1-null mice was recapitulated in IL-1b- and IL-18-knockout mice [55]. These results suggest that the activation of caspase-1 is required for the timely and adequate inflammatory response to the bacterial infection. If this hypothesis is correct, caspase-1induced inflammation might promote rather than limit the spread of bacteria in host tissue in the early phase of the infection. Alternatively, caspase-1-dependent apoptosis might facilitate bacterial spread from the intestines in another way. Certain immune cells, such as dendritic cells and polymorphonuclear neutrophils [56] that are attracted to the site of inflammation, might provide Salmonella with an intracellular niche and transportation from the Peyer’s patches to mesenteric lymphoid organs. Indeed, inflammatory signals produced by caspase-1, especially IL-1b, are potent stimuli for dendritic cell maturation and migration [57]. Hence, it is possible that the only way for bacterial transport to occur to lymph nodes is within dendritic cells that have recognized and engulfed apoptotic bodies from Salmonella-infected macrophages. The role of SPI2-dependent cell death in the pathogenesis of Salmonella infections is still unclear. Although the process in vitro is partially dependent on caspase-1, in vivo caspase-1 is only important intestinally – that is, in http://ticb.trends.com
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the SPI2-independent phase of the infection (Fig. 1) [53]. Perhaps Salmonella has developed an additional way to induce host cell death in order to enhance further spread of the pathogen after intracellular replication in macrophages of the spleen and liver. Recently, Salmonella-induced macrophage apoptosis was shown to enhance the processing and MHCI/MHCIIassisted presentation of Salmonella-derived antigens by dendritic cells [58]. A role for this pathway in the establishment of antimicrobial immunity warrants further studies. Concluding remarks Through the process of evolution, many pathogenic bacteria have become skilful cell manipulators. Studying the pathogenesis of Salmonella has revealed how this microbe exploits many host cell processes by injecting virulence proteins. These factors are produced in a strict temporal and spatial fashion in distinct host environments. The fact that the use of a single host factor (caspase-1) by a single bacterial virulence factor (SipB) affects the outcome of a Salmonella infection so fundamentally is remarkable. Several issues, however, need to be addressed. These include a better understanding of the exact molecular mechanisms by which SipB activates caspase-1. The signal-transduction events upstream of caspase-1 and the downstream apoptotic pathways are still poorly understood. The exact in vivo role of caspase-1mediated apoptosis in salmonellosis still needs to be better defined. The detailed characterization of interactions between Salmonella virulence proteins and caspase-1 has many implications for the understanding of the biology of this pathogen. Increased knowledge of the functions of caspase-1 might also provide novel types of therapeutic strategies to combat other conditions where caspase-1 is involved in pathogenesis. These include endotoxic shock and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) [59]. At the same time, the progress in understanding the bacterial effectors mimicking and modulating cell processes provides new tools for cell biologists to dissect eukaryotic cellular pathways.
Acknowledgements We gratefully acknowledge the contribution of Denise M. Monack, Ernesto Munoz-Elias, Ba¨rbel Raupach and David S.Weiss, for their thoughtful comments. We also thank Volker Brinkmann for the electron microscopy figure.
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34 Andrei, C. (1999) The secretory route of the leaderless protein interleukin 1beta involves exocytosis of endolysosome-related vesicles. Mol. Biol. Cell 10, 1463 – 1475 35 MacKenzie, A. (2001) Rapid secretion of interleukin-1beta by microvesicle shedding. Immunity 15, 825– 835 36 Martinon, F. et al. (2002) The inflammasome: a molecular platform triggering activation of inflammatory caspases and processing of proIL-beta. Mol. Cell 10, 417 – 426 37 Fesik, S.W. (2000) Insights into programmed cell death through structural biology. Cell 103, 273– 282 38 Acehan, D. et al. (2002) Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation. Mol. Cell 9, 423 – 432 39 Shi, Y. (2002) Mechanisms of caspase activation and inhibition during apoptosis. Mol. Cell 9, 459– 470 40 Inohara, N. and Nunez, G. (2001) The NOD: a signaling module that regulates apoptosis and host defense against pathogens. Oncogene 20, 6473– 6481 41 Imler, J.L. and Hoffmann, J.A. (2001) Toll receptors in innate immunity. Trends Cell Biol. 11, 304 – 311 42 Inohara, N. et al. (2001) Human Nod1 confers responsiveness to bacterial lipopolysaccharides. J. Biol. Chem. 276, 2551– 2554 43 Girardin, S.E., et al. Nod2 is a general sensor of peptidoglycan through muramyl dipeptide (MDP) detection. J. Biol. Chem. (in press) 44 Aliprantis, A.O. et al. (2000) The apoptotic signaling pathway activated by Toll-like receptor-2. EMBO J. 19, 3325 – 3336 45 Jesenberger, V. (2000) Salmonella-induced caspase-2 activation in macrophages: a novel mechanism in pathogen-mediated apoptosis. J. Exp. Med. 192, 1035 – 1046 46 van der Velden, A.W. et al. (2000) Salmonella pathogenicity island 1-independent induction of apoptosis in infected macrophages by Salmonella enterica serotype typhimurium. Infect. Immun. 68, 5702– 5709 47 Monack, D.M. et al. (2001) Salmonella pathogenicity island 2-dependent macrophage death is mediated in part by the host cysteine protease caspase-1. Cell. Microbiol 3, 825 – 837 48 van Engeland, M. (1996) A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture. Cytometry 24, 131– 139 49 Brennan, M.A. and Cookson, B.T. (2000) Salmonella induces macrophage death by caspase-1-dependent necrosis. Mol. Microbiol. 38, 31 – 40 50 Boise, L.H. and Collins, C.M. (2001) Salmonella-induced cell death: apoptosis, necrosis or programmed cell death? Trends Microbiol. 9, 64 – 67 51 Watson, P.R. (2000) Salmonella enterica serovars Typhimurium and Dublin can lyse macrophages by a mechanism distinct from apoptosis. Infect. Immun. 68, 3744 – 3747 52 Leist, M. and Ja¨a¨ttela¨, M. (2001) Four deaths and a funeral: from caspases to alternative mechanisms. Nat. Rev. Mol. Cell Biol. 2, 589– 598 53 Monack, D.M. (2000) Salmonella exploits caspase-1 to colonize Peyer’s patches in a murine typhoid model. J. Exp. Med. 192, 249 – 258 54 Galyov, E.E. et al. (1997) A secreted effector protein of Salmonella dublin is translocated into eukaryotic cells and mediates inflammation and fluid secretion in infected ileal mucosa. Mol. Microbiol. 25, 903– 912 55 Sansonetti, P.J. et al. (2000) Caspase-1 activation of IL-1beta and IL-18 are essential for Shigella flexneri-induced inflammation. Immunity 12, 581– 590 56 Wick, M.J. (2002) The role of dendritic cells during Salmonella infection. Curr. Opin. Immunol. 14, 437– 443 57 Cumberbatch, M. et al. (2001) Interleukin [IL]-18 induces Langerhans cell migration by a tumour necrosis factor-alpha- and IL-1betadependent mechanism. Immunology 102, 323 – 330 58 Yrlid, U. and Wick, M.J. (2000) Salmonella-induced apoptosis of infected macrophages results in presentation of a bacteria-encoded antigen after uptake by bystander dendritic cells. J. Exp. Med. 191, 613– 624 59 Li, M. et al. (2000) Functional role of caspase-1 and caspase-3 in an ALS transgenic mouse model. Science 288, 335 – 339
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Caspase-1 activation by Salmonella Harri A. Ja¨rvela¨inen, Antoine Galmiche and Arturo Zychlinsky Max Planck Institute for Infection Biology, Department of Cellular Microbiology, Campus Charite´ Mitte, Schumannstraße 21/22, Berlin 10117, Germany.
Salmonella is an interesting example of how the selective pressure of host environments has led to the evolution of sophisticated bacterial virulence mechanisms. This microbe exploits the first-line of defence, the macrophage, as a crucial tool in the initiation of disease. After invasion of intestinal macrophages, a virulence protein secreted by Salmonella specifically induces apoptotic cell death by activating the cysteine protease caspase-1. The pro-apoptotic capability is necessary for successful pathogenesis. The study of mechanisms by which Salmonella induces programmed cell death offers new insights into how pathogens cause disease and into general mechanisms of activation of the innate immune system. Apoptosis is a genetically programmed cell event, in which the dying cell participates actively in its own death. It is evolutionarily conserved and found in all multicellular organisms, where it is essential for normal development and homeostasis. Apoptotic cell death results from activation of a cascade of enzymes called caspases [1]. Caspases are cysteine proteases, each having a characteristic role in the apoptotic process. Upstream initiator caspases are synthesized as inactive zymogens that usually are activated through regulated protein – protein interactions. The downstream effector caspases are cleaved by the initiator caspase in order to generate a catalytically active enzyme. Apoptosis of host cells is observed in many different bacterial infections [2]. Depending on the bacteria, host cell apoptosis can be detrimental or advantageous to pathogenesis. Apoptosis often plays an important role in defence against intracellular pathogens, preventing the use of a safe niche within the host. For example, bronchial cell apoptosis mediated by the cell death receptor CD95 leads to increased survival of the host during murine Pseudomonas aeruginosa pneumonia, demonstrating the in vivo relevance of apoptosis in host defence against P. aeruginosa [3]. Salmonella, however, initiates apoptosis as a virulence strategy. Salmonellosis Salmonellae are Gram-negative facultative intracellular bacteria. Different Salmonella species cause a wide range of symptoms, ranging from a mild intestinal infection to life-threatening systemic infections. After ingestion, virulent Salmonella use principally M cells, specialized antigen-sampling epithelial cells lining the lymphoid Corresponding author: Arturo Zychlinsky ([email protected]).
follicles of the intestines, to cross the intestinal barrier (Fig. 1) [4,5]. Once inside the tissue, Salmonella infects macrophages, activates apoptosis and provokes mucosal inflammation. Bacteria are then transported via the lymphatics to mesenteric lymph nodes, which serve as a ‘distribution centre’ before Salmonella disseminates through the blood. Then, during several subsequent days, the bacteria reside and replicate intracellularly in phagocytic cells of the spleen and liver [6]. Salmonella pathogenesis can be studied with S. typhimurium infections in mice. Mice infected orally reproduce many, but not all, of the symptoms seen in the course of typhoid fever in humans [7,8]. Virulence mechanisms and intracellular life During cell invasion, Salmonella hijacks both the cytoskeleton and the membrane trafficking machinery of its target cells. At the site of cell contact, Salmonella effector proteins induce macropinocytic activities that change the membrane profoundly. The actin cytoskeleton rearrangements, which lead to Salmonella internalization, are achieved by the activation of host GTPases of the Rho family [9]. Salmonella survives and replicates within a unique vacuolar compartment called the ‘Salmonellacontaining vacuole’ derived from the late endosomal compartment of the cell [10,11]. Approximately 4% of the Salmonella genome, around 180 genes, is related to virulence. Many virulence genes are encoded in two large clusters within the Salmonella genome, Salmonella Pathogenicity Island (SPI) 1 and SPI2 [12 – 14]. These pathogenicity islands encode the components of two type III secretion apparatus, specialized secretion systems allowing the delivery of bacterial effector proteins inside the host cell [15]. Upon cell contact, this complex machinery is thought to form needle-like structures spanning the inner and outer membranes of the bacterial cell. Mutations in either one of these virulence islands lead to an attenuated infection in mice, but in different fashions (Fig. 1). Virulence genes in SPI1 are primarily expressed during the initial gastrointestinal phase of the infection. The SPI1 genes allow bacteria to infiltrate and establish infection within the epithelium and intestinal lymphoid tissues [16]. SPI1 is not necessary for the systemic phase of the infection as SPI1 mutants are not attenuated when they are administered intraperitoneally in mice, a model that bypasses the gastrointestinal phase. By contrast, genes encoded in SPI2 are necessary in the systemic phase of the infection as they enable the bacteria to survive and replicate within macrophages and other host cells [14,17].
http://ticb.trends.com 0962-8924/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0962-8924(03)00032-1
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Fig. 1. The natural history of a Salmonella infection. Genes encoded by the Salmonella Pathogenicity Island-1 and -2 (SPI1 and SPI2) have different roles during the course of Salmonella infection. The functions of SPI1 are necessary primarily during the intestinal phase of the infection (phase I), ranging from bacterial adherence and invasion to intracellular life in intestinal lymphoid tissues. The genes of SPI2 are crucial in the systemic phase of the disease (phase II), during which bacteria remain within macrophages in the spleen and liver.
Mutants in SPI2 are able to infect Peyer’s patches but fail to progress to the mesenteric lymph nodes. The early gastrointestinal phase of the infection is accompanied by the induction of acute mucosal inflammation [18]. This inflammation, as we will discuss later, requires the interaction of SPI1-encoded virulence protein Salmonella invasion protein B (SipB) with host cysteine protease caspase-1 [19]. SipB is required for the translocation of proteins through the type III secretion apparatus. Whether SipB is a ‘protein translocase’ in the membrane of host cells is still controversial [9,20]. SipB, however, plays an important intracellular role during the infection of eukaryotic cells. SipB is homologous (41% amino acid identity) and functionally analogous to the virulence factor IpaB produced by Shigella spp., a related organism that causes dysentery [21]. Although IpaB is thought to form pores in the plasma membrane of eukaryotic cells [22], neither pore formation nor hemolytic activity have been described for Salmonella. SipB is organized into two functional domains that probably account for the membrane-interaction properties. The amino-terminus might promote self-multimerization, whereas the carboxy-terminus interacts with membrane lipids [23]. Indeed, SipB appears to partition to membranes through its carboxy-terminal domain [23]. A topological analysis of SipB in phosphatidylcholine-containing liposomes suggests that SipB could span a complete bilayer [24]. It is still unclear whether this phenomenon is relevant for protein function in vivo. A comparative structure–function analysis of highly homologous regions of IpaB suggests that this might be the case [25]. A study linking the in vitro hydrophobic interactions of SipB and their role during Salmonella infections is warranted. Caspases Caspase-1 belongs to a mammalian family of caspases that consists of at least 14 members (Fig. 2) [26]. Each caspase http://ticb.trends.com
has a characteristic role in the initiation or execution of cell disassembly. Accordingly, they have been grouped into families known as ‘initiators’ and ‘executioners’. A diverse set of signals from outside and inside the cell can activate initiator caspases (including caspases-8, -9 and -10). Caspase-8 or -10-dependent pathways are triggered by their recruitment to the transmembrane domain of cellsurface death receptors (such as the TNF type 1 receptor family). Various forms of cellular stress, inducing p2
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Fig. 2. Phylogenetic classification of selected mammalian caspases (adapted from [39]). Effector caspases (such as caspase-3) contain no prodomain, whereas other members have either a death effector domain (DED, purple) or a CARD (caspase recruitment domain, yellow). CARD-containing caspases include caspase-1, caspase-5 (human) and caspase-11 (mouse). The catalytic domain of caspases shares significant structural homology among the various caspases: it contains a large 20-kDa and a small 10-kDa unit. Both of these subunits contribute four surface loops (L1– L4, blue) that shape the catalytic groove.
Functions of caspase-1 Caspase-1-dependent programmed cell death is distinct from other forms of ‘classical apoptosis’ as measured by at least three characteristics. First, the key elements of the mitochondrial pathway, such as caspase-3 and the proteins Bcl-2 and Bcl-xL, are not required in this cell death pathway [28]. Components of the caspase-1 apoptotic pathway are not known. One candidate could be raf-1, which is degraded during Salmonellainduced cell death, but the phenotype of raf-1-null macrophages is weak in Salmonella infections [29]. As caspase-3 is not involved in the caspase-1-dependent pathway, cleavage of poly (ADP-ribose) polymerase-1 (PARP-1) does not take place [18]. Second, in contrast to knockout mice of other caspases, which present with several developmental problems, caspase-1 knockout mice develop normally [30]. However, cells deficient in caspase-1 undergo apoptosis in response to many of the ‘classical’ apoptotic stimuli, such as UV irradiation or treatment with staurosporine [31]. Third, paradoxically, although apoptosis is thought to be an ‘immunologically silent’ process, caspase-1 is a proinflammatory enzyme. Caspase-1 cleaves the inactive precursors of IL-1b and IL-18, generating their mature active forms [32,33]. These cytokines do not contain the signal sequences of secretory proteins. It is still unclear how they are released from the cell after cleavage by caspase-1. Secretion of IL-1b might involve a nonclassical secretory pathway with exocytosis of lysosomal-related organelles [34] or plasma membrane vesiculation [35]. Alternatively, apoptosis itself might be required. Regulation of caspase-1 activation Protein – protein interactions most likely play a central part in the regulation of caspase-1. Martinon et al. [36] recently described that, in macrophages, caspase-1 is activated in a complex called the inflammasome. This large complex (more than 700 kDa) contains caspase-1 and caspase-5 as well as the adaptor proteins NALP1 and Pycard. These adaptors are essential in the assembly of the inflammasome. They contain either a Caspase Recruitment Domain (CARD) or the related Pyrin domain (PYD). These domains are found in the long prodomains of initiator caspases and belong to the death-domain-fold http://ticb.trends.com
superfamily, which includes the death domain (DD) and the death effector domain (DED) family [37]. Despite sharing little sequence identity, they fold into similar three-dimensional structures comprising six anti-parallel alpha helices. Both the CARD and PYD domains are able, like the DD and DED, to form homotypic interactions. The CARD or PYD domains interact highly specifically only with other CARD or PYD domains. In the case of the inflammasome, the CARD and PYD domains support the assembly of the complex by bringing together its protein components, including caspase-1 and caspase-5. The inflammasome is reminiscent of another caspase-activating complex of high molecular mass (1.4 MDa) – the apoptosome. The apoptosome drives the activation of caspase-9 following the release of cytochrome c from the mitochondrial intermembrane space. The resolution of the three-dimensional structure of the apoptosome revealed that it is a wheelshaped oligomer [38]. In addition to caspase-9, this complex contains the adaptor protein Apaf-1 (apoptotic protease-activating factor). In the presence of ATP or dATP, Apaf-1 binds to cytochrome c, triggering Apaf-1 selfmultimerization and formation of a platform on which caspase-9 docks and is activated [39]. Similarly to Apaf-1, NALP-1 contains a nucleotidebinding domain (NBD) (Fig. 3). This domain defines a family of factors, NODs (nucleotide-binding oligomerization
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cytochrome c release from the mitochondria, can initiate a ‘mitochondrial pathway’ leading to caspase-9 activation. Executioner caspases (such as caspase-3 and -7) are usually activated by proteolytic cleavage between domains by a previously activated upstream caspase. All caspases have three domains: an amino-terminal prodomain, and a catalytic domain, which consists of two subunits with shared structural homology among caspases (a large 20-kDa and a small 10-kDa unit). Cleavage usually occurs between the prodomain and the p20 domain after Asp residues. Therefore, the processing is believed to occur mostly autocatalytically. The affinity of each caspase for different substrates can vary considerably [27]. Ultimately, cell death takes place owing to the proteolytic cleavage of various cellular targets by activated caspases.
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Fig. 3. Regulatory proteins of caspase-1. Proteins listed have been documented to either activate or downregulate caspase-1 activity. The precursor of caspase-1 is activated by a proteolytic cleavage in the linker region between the amino-terminal CARD domain (yellow) and the two catalytic subunits (p20 and p10). Catalytic residues (including the cysteine at position 285) are represented here in purple. The CARD and pyrin (see text) domains are shown in green. These proteins use their pyrin or CARD domains to modulate the assembly of a caspase-1-activating platform, recently named the inflammasome (see text). A subset of adaptor proteins containing nucleotide-binding oligomerization domains (NODs), NALP1/CARD7, Ipaf/CARD12/Clan and Pypaf7, contain a centrally located nucleotide-binding site (NBS) in addition to repeated leucine-rich repeat sequences at their C-termini. Because these repeated sequences are homologous to those presumed to recognize bacterial structures in the Toll-like receptors (TLRs), it has been proposed that NOD proteins might function as cytoplasmic sensors for bacterial compounds.
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domains) that play a crucial role in caspase activation. NODs share a common structural organization, with an amino-terminal CARD/PYD domain, a centrally located NBD and repeated sequences at their carboxy-termini [40]. Interestingly, the leucine-rich repeats in NALP-1 are similar to those found in Toll-like receptors (TLRs), which recognize molecules common to many microbes [41]. Indeed, NODs appear to function as cytoplasmic sensors for microbial compounds [42,43]. Different microbial molecules and endogenous proinflammatory stimuli activate macrophages to release IL-1b. To what extent these proinflammatory stimuli induce apoptosis is not well characterized. An interesting case is the activation of TLRs, which under certain conditions can signal for apoptosis, and the release of IL-1b. In this case, apoptosis is dependent on caspase-8, but not caspase-1, whereas the release of mature IL-1b is dependent on caspase-1 [44]. Salmonella-induced cell death Salmonella-induced cell death requires caspase-1 activity as it is prevented by specific inhibitors (ValAla-Asp-fluoromethylketone; YVAD-FMK) and is not observed in macrophages derived from caspase-1knockout mice. A direct role for SipB in apoptosis is supported by the observation that microinjection of recombinant SipB protein is sufficient to induce apoptosis in macrophages [19]. However, the mechanism of caspase-1 activation by SipB is not clear. Binding studies performed with GST– SipB or immunoprecipitation experiments suggest that SipB binds to caspase-1 [19]. A direct contact could promote a change in caspase-1 conformation. Alternatively, SipB could help establish a local concentration of caspase-1 by favouring its auto-activation (a ‘nucleation’ effect) [39]. Interestingly, at high multiplicity of infection, cell death can be observed in caspase-1-deficient macrophages. Whether this represents a physiologically relevant pathway remains to be determined. It is possible that, in the absence of caspase-1, SipB can activate a closely related caspase [45]. Salmonella also induces SPI2-dependent, SPI1-independent apoptosis of macrophages [46,47]. This apoptosis is partly dependent on caspase-1 [47], but the bacterial effector protein has not yet been identified. In comparison with SipB-mediated apoptosis, the execution of SPI2-dependent cell death is considerably delayed (18 – 24 h post infection). Morphologically, Salmonella-induced cell death is characterized by classical apoptotic changes – formation of large cytoplasmic vacuoles, membrane blebs, chromatin condensation and cell shrinkage (Fig. 4) [18]. One parameter that has been used to characterize host cell death is the delay between the loss of phosphatidylserine (PtdSer) asymmetry in the plasma membrane and the subsequent destruction of membrane integrity [48]. Early apoptotic cells display PtdSer in their outer leaflet while maintaining membrane integrity. Monack et al. elegantly showed the kinetics of these steps using time-lapse video microscopy of Salmonella-infected macrophages: during infection of macrophages with Salmonella, binding of annexin-V to the PtdSer in the outer leaflet of the http://ticb.trends.com
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Fig. 4. Salmonella typhimurium-infected macrophages show classical apoptotic morphology. (a) Electron-microscopic features of apoptosis. Arrows show membrane blebbing and arrowheads show Salmonella-containing vacuoles. Chromatin condensation and margination can be seen in the nucleus. (b) The DNA-binding dye propidium iodide (red) allows the visualization of chromatin and intracellular bacteria. Following the induction of apoptosis, chromatin condensation occurs (globular shape in the nucleus). The dying cell exposes phosphatidylserine (PtdSer), an aminophospholipid normally restricted to the inner leaflet of the plasma membrane, on the cell surface. Annexin V (green) specifically binds to PtdSer [48].
plasma membrane precedes the ultimate loss of macrophage membrane integrity, although the interval between these two parameters is very short (approximately 2 minutes; see http://cmgm.stanford.edu/falkow/index.html). The fact that Salmonella-induced cell death exhibits unique features (see ‘functions of caspase-1’) not seen in ‘classical’ apoptosis has led some authors to compare it to another form of cell death, necrosis [49 – 51]. The aspects of distinctive features of Salmonella-induced cell death and their resemblance to necrosis have been recently discussed in detail [18]. The dogma that apoptosis is a single entity has recently been challenged. Clearly, there are many alternative pathways for the execution of programmed cell death, leading to varying apoptotic phenotypes [52].
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Caspase-1-mediated apoptosis in Salmonella infection in vivo The most convincing evidence for the crucial role of caspase-1 in salmonellosis comes from studies using knockout mice. A1000-fold higher dose of Salmonella is required to lethally infect caspase-1-knockout mice compared with wild-type mice [53]. In caspase-1-knockout mice, Salmonella infects the Peyer’s patch only transiently and it does not spread to mesenteric lymph nodes and spleen. However, when the mice are infected intraperitoneally, a route that bypasses the intestine, the caspase-1null mice do not present a phenotype [53]. Because these phenotypes are reminiscent of the one obtained after infection with SPI1 or sipB mutants [54], SipB has been suggested to be the main activator for caspase-1 during in vivo infection. However, it is difficult to directly address this possibility because of the multiple roles of SipB in addition to directly activating caspase-1. Although these in vivo studies clearly indicate that caspase-1 is required for the systemic spread of Salmonella from the Peyer’s patches, the functions of apoptosis are not clear. The most direct interpretation of the data is that, since macrophages can kill bacteria, in caspase-1null mice, the bacteria, rather than the macrophages, are killed. However, this might not be the case because the intracellular survival of Salmonella does not differ between wild-type and caspase-1-null macrophages in vitro [53]. It is possible that IL-1b and IL-18, the two cytokines activated by caspase-1, are necessary for the intestinal inflammation characteristic of salmonellosis. The inflammatory response might compromise the integrity of the intestinal barrier, resulting in a secondary bacterial invasion from the intestines. Indeed, in a mouse lung model of Shigella infection, the profound phenotype observed in infections of caspase-1-null mice was recapitulated in IL-1b- and IL-18-knockout mice [55]. These results suggest that the activation of caspase-1 is required for the timely and adequate inflammatory response to the bacterial infection. If this hypothesis is correct, caspase-1induced inflammation might promote rather than limit the spread of bacteria in host tissue in the early phase of the infection. Alternatively, caspase-1-dependent apoptosis might facilitate bacterial spread from the intestines in another way. Certain immune cells, such as dendritic cells and polymorphonuclear neutrophils [56] that are attracted to the site of inflammation, might provide Salmonella with an intracellular niche and transportation from the Peyer’s patches to mesenteric lymphoid organs. Indeed, inflammatory signals produced by caspase-1, especially IL-1b, are potent stimuli for dendritic cell maturation and migration [57]. Hence, it is possible that the only way for bacterial transport to occur to lymph nodes is within dendritic cells that have recognized and engulfed apoptotic bodies from Salmonella-infected macrophages. The role of SPI2-dependent cell death in the pathogenesis of Salmonella infections is still unclear. Although the process in vitro is partially dependent on caspase-1, in vivo caspase-1 is only important intestinally – that is, in http://ticb.trends.com
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the SPI2-independent phase of the infection (Fig. 1) [53]. Perhaps Salmonella has developed an additional way to induce host cell death in order to enhance further spread of the pathogen after intracellular replication in macrophages of the spleen and liver. Recently, Salmonella-induced macrophage apoptosis was shown to enhance the processing and MHCI/MHCIIassisted presentation of Salmonella-derived antigens by dendritic cells [58]. A role for this pathway in the establishment of antimicrobial immunity warrants further studies. Concluding remarks Through the process of evolution, many pathogenic bacteria have become skilful cell manipulators. Studying the pathogenesis of Salmonella has revealed how this microbe exploits many host cell processes by injecting virulence proteins. These factors are produced in a strict temporal and spatial fashion in distinct host environments. The fact that the use of a single host factor (caspase-1) by a single bacterial virulence factor (SipB) affects the outcome of a Salmonella infection so fundamentally is remarkable. Several issues, however, need to be addressed. These include a better understanding of the exact molecular mechanisms by which SipB activates caspase-1. The signal-transduction events upstream of caspase-1 and the downstream apoptotic pathways are still poorly understood. The exact in vivo role of caspase-1mediated apoptosis in salmonellosis still needs to be better defined. The detailed characterization of interactions between Salmonella virulence proteins and caspase-1 has many implications for the understanding of the biology of this pathogen. Increased knowledge of the functions of caspase-1 might also provide novel types of therapeutic strategies to combat other conditions where caspase-1 is involved in pathogenesis. These include endotoxic shock and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) [59]. At the same time, the progress in understanding the bacterial effectors mimicking and modulating cell processes provides new tools for cell biologists to dissect eukaryotic cellular pathways.
Acknowledgements We gratefully acknowledge the contribution of Denise M. Monack, Ernesto Munoz-Elias, Ba¨rbel Raupach and David S.Weiss, for their thoughtful comments. We also thank Volker Brinkmann for the electron microscopy figure.
References 1 Chang, H.Y. and Yang, X. (2000) Proteases for cell suicide: functions and regulation of caspases. Microbiol. Mol. Biol. Rev. 64, 821 – 846 2 Weinrauch, Y. and Zychlinsky, A. (1999) The induction of apoptosis by bacterial pathogens. Annu. Rev. Microbiol. 53, 155 – 187 3 Grassme, H. et al. (2000) CD95/CD95 ligand interactions on epithelial cells in host defense to Pseudomonas aeruginosa. Science 290, 527 – 530 4 Jones, B.D. et al. (1994) Salmonella typhimurium initiates murine infection by penetrating and destroying the specialized epithelial M cells of the Peyer’s patches. J. Exp. Med. 180, 15 – 23
Review
TRENDS in Cell Biology
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