- Email: [email protected]
Bacterial invasion: Force feeding by Salmonella
Dispatch
R277
Bacterial invasion: Force feeding by Salmonella John H. Brumell, Olivia Steele-Mortimer and B. Brett Finlay
Recent findings have shed new light on mammalian-cell invasion by Salmonella. Using a type III secretion system, Salmonella deliver virulence factors into the host cell that directly activate signal transduction pathways, initiating cytoskeletal rearrangements and bacterial uptake by a ruffling mechanism. Address: Biotechnology Laboratory, University of British Columbia, Vancouver, Canada. E-mail: [email protected] Current Biology 1999, 9:R277–R280 http://biomednet.com/elecref/09609822009R0277 © Elsevier Science Ltd ISSN 0960-9822
Invasive pathogens enter host cells for a number of reasons: to gain access to a replicative niche, evade the host immune system and also, in the case of Salmonella species, for delivery across the intestinal epithelium to underlying tissues. Depending on the pathogenic strain and the infected host, Salmonella infections can cause diseases ranging from a limited gastroenteritis (food poisoning) to systemic, often fatal, typhoid fever. In the latter case, colonization of host macrophages plays a critical role in progression of the disease [1]. Before host-adapted Salmonella can colonize macrophages, they must first cross the intestinal epithelium. By doing so, the bacteria enter their target epithelial cells by a mechanism quite distinct from receptor-mediated phagocytosis, or ‘cell eating’. Unlike professional phagocytes, such as neutrophils and macrophages, the targets of Salmonella invasion in the gut do not express specific phagocytic receptors for their uptake. Instead, Salmonella rely on their own invasive strategy which involves delivery of virulence factors to the host cytosol and induction of ruffling on the host cell plasma membrane, leading to their uptake. In this way, Salmonella are able to ‘force feed’ themselves to host cells of the epithelium, providing a replicative niche for the bacteria and access to underlying tissues. A number of recent findings have provided new insights into the mechanism and consequences of the delivery of Salmonella virulence factors into host cells. The Salmonella invasion process
Studies of Salmonella invasion have been done mainly with S. typhimurium, a strain which causes a systemic disease in mice that resembles typhoid fever in humans. S. typhimurium cells preferentially attach to, and invade, M cells of the ileal Peyers’ patches by a ruffle-mediated mechanism similar to that seen in vitro with epithelial cell lines. Invasion is initiated by activation of host signal
transduction cascades (see below) and dependent on rearrangement of the actin cytoskeleton. Major changes are induced in the host plasma membrane, including localized membrane ruffling and macropinocytosis. In some cases, the membrane ruffles resemble a large ‘splash’ on the surface of the host cell, like a stone hitting water. The invading bacteria are taken up in large vacuoles, morphologically similar to macropinosomes formed following growth factor stimulation, and non-invasive bacteria or latex beads can also be co-internalized. Receptor-mediated phagocytosis is morphologically quite distinct from the Salmonella invasion process and highly specific, entailing a progressive ‘zippering’ of the plasma membrane around receptor-bound particles and internalization into a tight-fitting phagosome. Virulence factors
Early in their evolution, Salmonella species acquired a cluster of chromosomal genes required for infection of animals. This region, the Salmonella Pathogenicity Island-1 (SPI-1) contains more than 28 genes that encode a complex bacterial type III secretion system, as well as secreted proteins and regulatory components [2]. Bacterial type III secretion systems are central to the virulence of many Gram-negative bacterial pathogens of plants and animals. Mutants of S. typhimurium defective for secretion through the SPI-1 type III system are unable to invade cultured epithelial cells in vitro and are attenuated for virulence in orally innoculated mice. Electron microscopy of S. typhimurium has revealed needlelike complexes spanning the inner and outer bacterial membranes [3]. These complexes have been purified by CsCl density gradient centrifugation and found to contain several components of the SPI-1 type III secretion system. Epitope tagging of the identified SPI-1 components enabled the purified needles to be visualized by immunoelectron microscopy. The SPI-1 type III secretion system thus forms a supramolecular structure across the bacterial envelope and appears to be directly involved in delivery of virulence factors into the host cell for invasion [3]. The translocation of virulence factors into host cells, via the SPI-1 secretion system, enables Salmonella cells to initiate a wide variety of intracellular signaling pathways, leading to membrane ruffling and uptake by the host cells (Figure 1). Recent studies have identified novel virulence factors which, though not encoded within the SPI-1 chromosomal region, are nonetheless translocated into host cells via this type III secretion system. The targets of the translocated virulence factors, and the host
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Figure 1 Activation of host signaling pathways in Salmonella host cells via delivery of SPI-1 virulence factors (yellow boxes). ERK, extracellular regulated kinase; JNK, c-Jun N-terminal kinase; SPI-1, Salmonella Pathogenicity Island-1. (See text for details.)
Salmonella
Host cell cytosol SopE
SptP
SipA
SopB
SipB, SipC, others?
GTP GDP Rho GTPases
ERK/JNK/p38
Actin rearrangements
Lipid dephosphorylation
?
Cytokine production
cell responses initiated by their interactions, are the subject of intensive investigation.
?
SPI-1 type III secretion system Current Biology
another protein, T-plastin. S. typhimurium thus possess several virulence factors that manipulate the host actin cytoskeleton by multiple mechanisms.
Actin rearrangements
Rho family GTPases regulate numerous cellular events, including polymerization of actin and initiation of mitogen activated protein (MAP) kinase cascades. SopE, a translocated effector of the SPI-1 secretion system in S. typhimurium — though not itself encoded within SPI-1 — is a guanine-nucleotide exchange factor for some Rho GTPases, including Cdc42 and Rac which are required for invasion [4]. Surprisingly, SopE does not show any obvious amino-acid sequence similarity to other known regulators of small GTPases. Within the S. typhimurium SPI-1 region is a gene encoding a modular protein, SptP, that contains an amino-terminal region with homology to two other bacterial toxins — exotoxin S from Pseudomonas aeruginosa and YopE from Yersinia spp — followed by a protein tyrosine phosphatase domain that is catalytically competent in vitro [5]. Microinjection of either domain into epithelial cells disrupts the actin cytoskeleton, suggesting that the two domains cooperate to exert related functions upon translocation into host cells [6]. The host cell targets of both SptP domains remain to be determined. The protein SipA, also encoded within the SPI-1 region, is required for efficient internalization of S. typhimurium by epithelial cells, and is recruited to bacterial-induced ruffles at the host plasma membrane [7]. SipA binds actin, causing an increase in the actin-bundling activity of
Lipid dephosphorylation
SopB, encoded within a separate pathogenicity island (SPI-5) of the closely related S. dublin, is also translocated into host cells by the SPI-1 secretion system. While not essential for invasion of the intestinal epithelium, SopB is required for the induction of fluid secretion and neutrophil influx in a calf ileal-loop infection model [8]. SopB is an inositol phosphate phosphatase that hydrolyzes a wide variety of phosphoinositides, including phosphatidylinositol 3,4,5-trisphosphate (PIP3), an inhibitor of Ca2+-dependent chloride secretion [9]. SopB activity also leads to the generation of inositol 1,4,5,6-tetrakisphosphate, a signaling molecule that increases chloride secretion indirectly by antagonizing the actions of PIP3 [10]. S. typhimurium also produce a homologue of SopB, known as SigD, which is 98% identical in sequence to SopB and is necessary for efficient invasion of epithelial cells [11]. How SigD might influence S. typhimurium invasion by manipulating phosphoinositide signaling remains unclear. These findings illustrate how Salmonella species mediate their virulence through the modification of both protein and lipid signaling pathways in host cells. Other pathways?
SipB and SipC are essential for Salmonella invasion, and are directly involved in the delivery of secreted proteins into the host cell. SipB and SipC are themselves similarly
Dispatch
translocated into the host cell, suggesting they might also function as effector molecules [12]. Support for this notion comes from the recent finding that expression at the plasma membrane of epithelial cells of receptor chimeras bearing the carboxyl terminus of SipB or the amino terminus of SipC blocks S. typhimurium invasion [13]. The host cell signaling pathways affected by these virulence factors remain unknown. From the brief descriptions above, it is clear that much remains to be determined about the mechanisms by which the S. typhimurium virulence factors collectively mediate invasion. Furthermore, Salmonella species secrete a large number of proteins and it is expected that other translocated virulence factors will be identified. Some of these may be species-specific, as is SopE, providing a unique arsenal of secreted effector molecules to infect the host that each species has adapted to. Studies of S. typhimurium have shown how a variety of virulence factors can activate a single, but obligatory, host-cell response — such as actin rearrangements — by different mechanisms. This overlapping of functions helps to explain why mutations of some virulence factors have only a slight effect on S. typhimurium invasion. Further study of the secreted effectors that Salmonella species use for host-cell invasion should provide insight into both pathogenesis of these bacteria and molecular mechanisms controlling endocytosis. Invasion versus phagocytosis
The recent discovery and characterization of the virulence factors described above has emphasized the unique and aggressive nature of S. typhimurium invasion. Table 1 shows a comparison of S. typhimurium invasion with receptor-mediated phagocytosis by professional phagocytes. It is particularly notable that in vitro invasion by S. typhimurium is dependent on many factors, such as growth phase, oxygen and osmolarity. Receptor-mediated phagocytosis, by contrast, is independent of the ingested particle and requires only efficient recognition by cell surface receptors. While S. typhimurium surface determinants exist for selective binding to M cells, no host-cell surface receptor has been identified for uptake [14]. The initial bacteria–epithelium interaction must thus be followed by translocation of virulence factors into the host cell for invasion to occur. A number of important differences have been identified between the signaling requirements for phagocytosis, on the one hand, and S. typhimurium invasion, on the other (Table 1). Both require inositol (1,4,5)-trisphosphate (IP3) and Ca2+ transients and activation of Rho GTPases. S. typhimurium invasion, however, is independent of tyrosine kinases [15] and phosphatidylinositol 3-kinases [16], both of which are necessary for receptor-mediated phagocytosis to occur.
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Table 1 Comparison of phagocytosis and Salmonella invasion. Phagocytosis
Salmonella invasion
Particle
Inert particle; must have surface ligand for recognition by receptors
Bacteria must be viable; many factors affect invasion
Receptors
Mediate adherence and uptake; bind immunoglobulin, mannose, complement and so on
None identified for uptake
Obligatory signals IP3/Ca2+; PTK; PKC PI 3-K, Rho GTPases
IP3/Ca2+, Cdc42/Rac
Cytoskeleton
Actin polymerization required
Actin polymerization required
Kinetics
Fast (minutes)
Fast (minutes) if bacteria invasive
Morphology
‘Zippers’
‘Big gulp’ or ‘splash’
Initial vacuole
Tight fitting around particle
Spacious
Intracellular transport
Follows normal endocytic route; delivery of lysosomal enzymes
Uncoupled from endocytic route; lack CI-M6PR, cathepsins D/L
Vacuolar pH
Rapid acidification mediated by delivery of vacuolar type H+-ATPase
Acidification, but may be slower, less acidic than usual; necessary for gene induction
CI-M6PR, calcium-independent mannose 6-phosphate receptor; PKC, protein kinase C; PI 3-K, phosphoinositide inositol 3-kinase; PTK, protein tyrosine kinase.
Survival in the host cell
The intracellular transport of S. typhimurium-containing vesicles is also quite distinct from that of particles taken up by receptor-mediated phagocytosis (Table 1). Unlike vesicles containing dead or non-virulent S. typhimurium strains, vacuoles containing live bacteria do not acquire mannose 6-phosphate receptors or cathepsins D and L. These findings were previously thought limited to the transport of S. typhimurium-containing vesicles in epithelial cells, but they have recently been extended to macrophages by immunofluorescence analysis [17] and purification of S. typhimurium-containing vesicles by subcellular fractionation [18]. It is thought that, by uncoupling themselves from the normal intracellular transport route, the bacteria avoid exposure to harsh lysosomal agents, increasing their changes of survival and replication. Interestingly, a noninvasive SPI-1 mutant of S. typhimurium becomes localized in an intracellular compartment identical to that of wildtype bacteria in macrophages after ingestion by receptormediated phagocytosis [17]. This suggests that S. typhimurium is capable of directing its intracellular
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Current Biology, Vol 9 No 8
transport regardless of the method of uptake by macrophages (invasion or phagocytosis). These findings further suggest that SPI-1-translocated virulence factors do not mediate alteration of the endocytic pathway. Such alterations are likely performed by other virulence factors, possibly those encoded within four separate pathogenicity ‘islands’ or the many related pathogenicity ‘islets’ that have been identified to date [2]. The details of how these, and perhaps other, virulence factors affect host cell machinery remain to be determined. Concluding remarks
Salmonella species clearly manipulate signal transduction pathways in their host mammalian cells for their own ends. Through the actions of virulence factors delivered via a conserved type III secretion system, they can induce their own uptake into cells. Once inside the host cell, they uncouple normal intracellular transport of the Salmonellacontaining vacuole to promote their survival and replication. It is hoped that future studies of the translocated S. typhimurium virulence factors and their host-cell targets will elucidate the key mechanisms that regulate this specialized endocytic pathway. Acknowledgements Work in B.B.F.’s laboratory is supported by operating grants from the Medical Research Council of Canada, the Canadian Bacterial Diseases Network, and the Natural Sciences and Engineering Research Council of Canada. J.H.B. is supported by a postdoctoral fellowship from NSERC, and B.B.F. is a Howard Hughes International Scholar and an MRC Scientist.
References 1. Jones BD, Falkow S: Salmonellosis: host immune responses and bacterial virulence determinants. Annu Rev Immunol 1996, 14:533-561. 2. Collazo CM, Galan JE: The invasion-associated type-III protein secretion system in Salmonella - a review. Gene 1997, 192:51-59. 3. Kubori T, Matsushima Y, Nakamura D, Uralil J, Lara-Tejero M, Sukhan A, Galan JE, Aizawa SI: Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science 1998, 280:602-605. 4. Hardt WD, Chen LM, Schuebel KE, Bustelo XR, Galan JE: S. typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells. Cell 1998, 93:815-826. 5. Kaniga K, Uralil J, Bliska JB, Galan JE: A secreted protein tyrosine phosphatase with modular effector domains in the bacterial pathogen Salmonella typhimurium. Mol Microbiol 1996, 21:633-641. 6. Fu Y, Galan JE: The Salmonella typhimurium tyrosine phosphatase SptP is translocated into host cells and disrupts the actin cytoskeleton. Mol Microbiol 1998, 27:359-368. 7. Zhou D, Galan JE: S. typhimurium encodes an actin-binding protein that modulates the bundling activity of plastin to promote bacterial entry into host cells. In Annual Meeting of the American Society for Cell Biology. Mol Biol Cell 1998, 9 Suppl:2240 8. Galyov EE, Wood MW, Rosqvist R, Mullan PB, Watson PR, Hedges S, Wallis TS: A secreted effector protein of Salmonella dublin is translocated into eukaryotic cells and mediates inflammation and fluid secretion in infected ileal mucosa. Mol Microbiol 1997, 25:903-912. 9. Norris FA, Wilson MP, Wallis TS, Galyov EE, Majerus PW: SopB, a protein required for virulence of Salmonella dublin, is an inositol phosphate phosphatase. Proc Natl Acad Sci USA 1998, 95:14057-14059.
10. Eckmann L, Rudolf MT, Ptasznik A, Schultz C, Jiang T, Wolfson N, Tsien R, Fierer J, Shears SB, Kagnoff MF et al.: D-myo-inositol 1,4,5,6-tetrakisphosphate produced in human intestinal epithelial cells in response to Salmonella invasion inhibits phosphoinositide 3-kinase signaling pathways. Proc Natl Acad Sci USA 1997, 94:14456-14460. 11. Hong KH, Miller VL: Identification of a novel Salmonella invasion locus homologous to Shigella ipgDE. J Bacteriol 1998, 180:1793-1802. 12. Collazo CM, Galan JE: The invasion-associated type III system of Salmonella typhimurium directs the translocation of Sip proteins into the host cell. Mol Microbiol 1997, 24:747-756. 13. Carlson SA, Jones BD: Inhibition of Salmonella typhimurium invasion by host cell expression of secreted bacterial invasion proteins. Infect Immun 1998, 66:5295-5300. 14. Jones B, Pascopella L, Falkow S: Entry of microbes into the host: using M cells to break the mucosal barrier. Curr Opin Immunol 1995, 7:474-478. 15. Rosenshine I, Ruschkowski S, Foubister V, Finlay BB: Salmonella typhimurium invasion of epithelial cells: role of induced host cell tyrosine protein phosphorylation. Infect Immun 1994, 62:4969-4974. 16. Mecsas J, Raupach B, Falkow S: The Yersinia Yops inhibit invasion of Listeria, Shigella and Edwardsiella but not Salmonella into epithelial cells. Mol Microbiol 1998, 28:1269-1281. 17. Rathman M, Barker LP, Falkow S: The unique trafficking pattern of Salmonella typhimurium-containing phagosomes in murine macrophages is independent of the mechanism of bacterial entry. Infect Immun 1997, 65:1475-1485. 18. Mills SD, Finlay BB: Isolation and characterization of Salmonella typhimurium and Yersinia pseudotuberculosis-containing phagosomes from infected mouse macrophages: Y. pseudotuberculosis traffics to terminal lysosomes where they are degraded. Eur J Cell Biol 1998, 77:35-47.
R277
Bacterial invasion: Force feeding by Salmonella John H. Brumell, Olivia Steele-Mortimer and B. Brett Finlay
Recent findings have shed new light on mammalian-cell invasion by Salmonella. Using a type III secretion system, Salmonella deliver virulence factors into the host cell that directly activate signal transduction pathways, initiating cytoskeletal rearrangements and bacterial uptake by a ruffling mechanism. Address: Biotechnology Laboratory, University of British Columbia, Vancouver, Canada. E-mail: [email protected] Current Biology 1999, 9:R277–R280 http://biomednet.com/elecref/09609822009R0277 © Elsevier Science Ltd ISSN 0960-9822
Invasive pathogens enter host cells for a number of reasons: to gain access to a replicative niche, evade the host immune system and also, in the case of Salmonella species, for delivery across the intestinal epithelium to underlying tissues. Depending on the pathogenic strain and the infected host, Salmonella infections can cause diseases ranging from a limited gastroenteritis (food poisoning) to systemic, often fatal, typhoid fever. In the latter case, colonization of host macrophages plays a critical role in progression of the disease [1]. Before host-adapted Salmonella can colonize macrophages, they must first cross the intestinal epithelium. By doing so, the bacteria enter their target epithelial cells by a mechanism quite distinct from receptor-mediated phagocytosis, or ‘cell eating’. Unlike professional phagocytes, such as neutrophils and macrophages, the targets of Salmonella invasion in the gut do not express specific phagocytic receptors for their uptake. Instead, Salmonella rely on their own invasive strategy which involves delivery of virulence factors to the host cytosol and induction of ruffling on the host cell plasma membrane, leading to their uptake. In this way, Salmonella are able to ‘force feed’ themselves to host cells of the epithelium, providing a replicative niche for the bacteria and access to underlying tissues. A number of recent findings have provided new insights into the mechanism and consequences of the delivery of Salmonella virulence factors into host cells. The Salmonella invasion process
Studies of Salmonella invasion have been done mainly with S. typhimurium, a strain which causes a systemic disease in mice that resembles typhoid fever in humans. S. typhimurium cells preferentially attach to, and invade, M cells of the ileal Peyers’ patches by a ruffle-mediated mechanism similar to that seen in vitro with epithelial cell lines. Invasion is initiated by activation of host signal
transduction cascades (see below) and dependent on rearrangement of the actin cytoskeleton. Major changes are induced in the host plasma membrane, including localized membrane ruffling and macropinocytosis. In some cases, the membrane ruffles resemble a large ‘splash’ on the surface of the host cell, like a stone hitting water. The invading bacteria are taken up in large vacuoles, morphologically similar to macropinosomes formed following growth factor stimulation, and non-invasive bacteria or latex beads can also be co-internalized. Receptor-mediated phagocytosis is morphologically quite distinct from the Salmonella invasion process and highly specific, entailing a progressive ‘zippering’ of the plasma membrane around receptor-bound particles and internalization into a tight-fitting phagosome. Virulence factors
Early in their evolution, Salmonella species acquired a cluster of chromosomal genes required for infection of animals. This region, the Salmonella Pathogenicity Island-1 (SPI-1) contains more than 28 genes that encode a complex bacterial type III secretion system, as well as secreted proteins and regulatory components [2]. Bacterial type III secretion systems are central to the virulence of many Gram-negative bacterial pathogens of plants and animals. Mutants of S. typhimurium defective for secretion through the SPI-1 type III system are unable to invade cultured epithelial cells in vitro and are attenuated for virulence in orally innoculated mice. Electron microscopy of S. typhimurium has revealed needlelike complexes spanning the inner and outer bacterial membranes [3]. These complexes have been purified by CsCl density gradient centrifugation and found to contain several components of the SPI-1 type III secretion system. Epitope tagging of the identified SPI-1 components enabled the purified needles to be visualized by immunoelectron microscopy. The SPI-1 type III secretion system thus forms a supramolecular structure across the bacterial envelope and appears to be directly involved in delivery of virulence factors into the host cell for invasion [3]. The translocation of virulence factors into host cells, via the SPI-1 secretion system, enables Salmonella cells to initiate a wide variety of intracellular signaling pathways, leading to membrane ruffling and uptake by the host cells (Figure 1). Recent studies have identified novel virulence factors which, though not encoded within the SPI-1 chromosomal region, are nonetheless translocated into host cells via this type III secretion system. The targets of the translocated virulence factors, and the host
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Current Biology, Vol 9 No 8
Figure 1 Activation of host signaling pathways in Salmonella host cells via delivery of SPI-1 virulence factors (yellow boxes). ERK, extracellular regulated kinase; JNK, c-Jun N-terminal kinase; SPI-1, Salmonella Pathogenicity Island-1. (See text for details.)
Salmonella
Host cell cytosol SopE
SptP
SipA
SopB
SipB, SipC, others?
GTP GDP Rho GTPases
ERK/JNK/p38
Actin rearrangements
Lipid dephosphorylation
?
Cytokine production
cell responses initiated by their interactions, are the subject of intensive investigation.
?
SPI-1 type III secretion system Current Biology
another protein, T-plastin. S. typhimurium thus possess several virulence factors that manipulate the host actin cytoskeleton by multiple mechanisms.
Actin rearrangements
Rho family GTPases regulate numerous cellular events, including polymerization of actin and initiation of mitogen activated protein (MAP) kinase cascades. SopE, a translocated effector of the SPI-1 secretion system in S. typhimurium — though not itself encoded within SPI-1 — is a guanine-nucleotide exchange factor for some Rho GTPases, including Cdc42 and Rac which are required for invasion [4]. Surprisingly, SopE does not show any obvious amino-acid sequence similarity to other known regulators of small GTPases. Within the S. typhimurium SPI-1 region is a gene encoding a modular protein, SptP, that contains an amino-terminal region with homology to two other bacterial toxins — exotoxin S from Pseudomonas aeruginosa and YopE from Yersinia spp — followed by a protein tyrosine phosphatase domain that is catalytically competent in vitro [5]. Microinjection of either domain into epithelial cells disrupts the actin cytoskeleton, suggesting that the two domains cooperate to exert related functions upon translocation into host cells [6]. The host cell targets of both SptP domains remain to be determined. The protein SipA, also encoded within the SPI-1 region, is required for efficient internalization of S. typhimurium by epithelial cells, and is recruited to bacterial-induced ruffles at the host plasma membrane [7]. SipA binds actin, causing an increase in the actin-bundling activity of
Lipid dephosphorylation
SopB, encoded within a separate pathogenicity island (SPI-5) of the closely related S. dublin, is also translocated into host cells by the SPI-1 secretion system. While not essential for invasion of the intestinal epithelium, SopB is required for the induction of fluid secretion and neutrophil influx in a calf ileal-loop infection model [8]. SopB is an inositol phosphate phosphatase that hydrolyzes a wide variety of phosphoinositides, including phosphatidylinositol 3,4,5-trisphosphate (PIP3), an inhibitor of Ca2+-dependent chloride secretion [9]. SopB activity also leads to the generation of inositol 1,4,5,6-tetrakisphosphate, a signaling molecule that increases chloride secretion indirectly by antagonizing the actions of PIP3 [10]. S. typhimurium also produce a homologue of SopB, known as SigD, which is 98% identical in sequence to SopB and is necessary for efficient invasion of epithelial cells [11]. How SigD might influence S. typhimurium invasion by manipulating phosphoinositide signaling remains unclear. These findings illustrate how Salmonella species mediate their virulence through the modification of both protein and lipid signaling pathways in host cells. Other pathways?
SipB and SipC are essential for Salmonella invasion, and are directly involved in the delivery of secreted proteins into the host cell. SipB and SipC are themselves similarly
Dispatch
translocated into the host cell, suggesting they might also function as effector molecules [12]. Support for this notion comes from the recent finding that expression at the plasma membrane of epithelial cells of receptor chimeras bearing the carboxyl terminus of SipB or the amino terminus of SipC blocks S. typhimurium invasion [13]. The host cell signaling pathways affected by these virulence factors remain unknown. From the brief descriptions above, it is clear that much remains to be determined about the mechanisms by which the S. typhimurium virulence factors collectively mediate invasion. Furthermore, Salmonella species secrete a large number of proteins and it is expected that other translocated virulence factors will be identified. Some of these may be species-specific, as is SopE, providing a unique arsenal of secreted effector molecules to infect the host that each species has adapted to. Studies of S. typhimurium have shown how a variety of virulence factors can activate a single, but obligatory, host-cell response — such as actin rearrangements — by different mechanisms. This overlapping of functions helps to explain why mutations of some virulence factors have only a slight effect on S. typhimurium invasion. Further study of the secreted effectors that Salmonella species use for host-cell invasion should provide insight into both pathogenesis of these bacteria and molecular mechanisms controlling endocytosis. Invasion versus phagocytosis
The recent discovery and characterization of the virulence factors described above has emphasized the unique and aggressive nature of S. typhimurium invasion. Table 1 shows a comparison of S. typhimurium invasion with receptor-mediated phagocytosis by professional phagocytes. It is particularly notable that in vitro invasion by S. typhimurium is dependent on many factors, such as growth phase, oxygen and osmolarity. Receptor-mediated phagocytosis, by contrast, is independent of the ingested particle and requires only efficient recognition by cell surface receptors. While S. typhimurium surface determinants exist for selective binding to M cells, no host-cell surface receptor has been identified for uptake [14]. The initial bacteria–epithelium interaction must thus be followed by translocation of virulence factors into the host cell for invasion to occur. A number of important differences have been identified between the signaling requirements for phagocytosis, on the one hand, and S. typhimurium invasion, on the other (Table 1). Both require inositol (1,4,5)-trisphosphate (IP3) and Ca2+ transients and activation of Rho GTPases. S. typhimurium invasion, however, is independent of tyrosine kinases [15] and phosphatidylinositol 3-kinases [16], both of which are necessary for receptor-mediated phagocytosis to occur.
R279
Table 1 Comparison of phagocytosis and Salmonella invasion. Phagocytosis
Salmonella invasion
Particle
Inert particle; must have surface ligand for recognition by receptors
Bacteria must be viable; many factors affect invasion
Receptors
Mediate adherence and uptake; bind immunoglobulin, mannose, complement and so on
None identified for uptake
Obligatory signals IP3/Ca2+; PTK; PKC PI 3-K, Rho GTPases
IP3/Ca2+, Cdc42/Rac
Cytoskeleton
Actin polymerization required
Actin polymerization required
Kinetics
Fast (minutes)
Fast (minutes) if bacteria invasive
Morphology
‘Zippers’
‘Big gulp’ or ‘splash’
Initial vacuole
Tight fitting around particle
Spacious
Intracellular transport
Follows normal endocytic route; delivery of lysosomal enzymes
Uncoupled from endocytic route; lack CI-M6PR, cathepsins D/L
Vacuolar pH
Rapid acidification mediated by delivery of vacuolar type H+-ATPase
Acidification, but may be slower, less acidic than usual; necessary for gene induction
CI-M6PR, calcium-independent mannose 6-phosphate receptor; PKC, protein kinase C; PI 3-K, phosphoinositide inositol 3-kinase; PTK, protein tyrosine kinase.
Survival in the host cell
The intracellular transport of S. typhimurium-containing vesicles is also quite distinct from that of particles taken up by receptor-mediated phagocytosis (Table 1). Unlike vesicles containing dead or non-virulent S. typhimurium strains, vacuoles containing live bacteria do not acquire mannose 6-phosphate receptors or cathepsins D and L. These findings were previously thought limited to the transport of S. typhimurium-containing vesicles in epithelial cells, but they have recently been extended to macrophages by immunofluorescence analysis [17] and purification of S. typhimurium-containing vesicles by subcellular fractionation [18]. It is thought that, by uncoupling themselves from the normal intracellular transport route, the bacteria avoid exposure to harsh lysosomal agents, increasing their changes of survival and replication. Interestingly, a noninvasive SPI-1 mutant of S. typhimurium becomes localized in an intracellular compartment identical to that of wildtype bacteria in macrophages after ingestion by receptormediated phagocytosis [17]. This suggests that S. typhimurium is capable of directing its intracellular
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Current Biology, Vol 9 No 8
transport regardless of the method of uptake by macrophages (invasion or phagocytosis). These findings further suggest that SPI-1-translocated virulence factors do not mediate alteration of the endocytic pathway. Such alterations are likely performed by other virulence factors, possibly those encoded within four separate pathogenicity ‘islands’ or the many related pathogenicity ‘islets’ that have been identified to date [2]. The details of how these, and perhaps other, virulence factors affect host cell machinery remain to be determined. Concluding remarks
Salmonella species clearly manipulate signal transduction pathways in their host mammalian cells for their own ends. Through the actions of virulence factors delivered via a conserved type III secretion system, they can induce their own uptake into cells. Once inside the host cell, they uncouple normal intracellular transport of the Salmonellacontaining vacuole to promote their survival and replication. It is hoped that future studies of the translocated S. typhimurium virulence factors and their host-cell targets will elucidate the key mechanisms that regulate this specialized endocytic pathway. Acknowledgements Work in B.B.F.’s laboratory is supported by operating grants from the Medical Research Council of Canada, the Canadian Bacterial Diseases Network, and the Natural Sciences and Engineering Research Council of Canada. J.H.B. is supported by a postdoctoral fellowship from NSERC, and B.B.F. is a Howard Hughes International Scholar and an MRC Scientist.
References 1. Jones BD, Falkow S: Salmonellosis: host immune responses and bacterial virulence determinants. Annu Rev Immunol 1996, 14:533-561. 2. Collazo CM, Galan JE: The invasion-associated type-III protein secretion system in Salmonella - a review. Gene 1997, 192:51-59. 3. Kubori T, Matsushima Y, Nakamura D, Uralil J, Lara-Tejero M, Sukhan A, Galan JE, Aizawa SI: Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science 1998, 280:602-605. 4. Hardt WD, Chen LM, Schuebel KE, Bustelo XR, Galan JE: S. typhimurium encodes an activator of Rho GTPases that induces membrane ruffling and nuclear responses in host cells. Cell 1998, 93:815-826. 5. Kaniga K, Uralil J, Bliska JB, Galan JE: A secreted protein tyrosine phosphatase with modular effector domains in the bacterial pathogen Salmonella typhimurium. Mol Microbiol 1996, 21:633-641. 6. Fu Y, Galan JE: The Salmonella typhimurium tyrosine phosphatase SptP is translocated into host cells and disrupts the actin cytoskeleton. Mol Microbiol 1998, 27:359-368. 7. Zhou D, Galan JE: S. typhimurium encodes an actin-binding protein that modulates the bundling activity of plastin to promote bacterial entry into host cells. In Annual Meeting of the American Society for Cell Biology. Mol Biol Cell 1998, 9 Suppl:2240 8. Galyov EE, Wood MW, Rosqvist R, Mullan PB, Watson PR, Hedges S, Wallis TS: A secreted effector protein of Salmonella dublin is translocated into eukaryotic cells and mediates inflammation and fluid secretion in infected ileal mucosa. Mol Microbiol 1997, 25:903-912. 9. Norris FA, Wilson MP, Wallis TS, Galyov EE, Majerus PW: SopB, a protein required for virulence of Salmonella dublin, is an inositol phosphate phosphatase. Proc Natl Acad Sci USA 1998, 95:14057-14059.
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