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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. General features of SPI-2
  5. Physiological activities of the SPI-2 TTSS
  6. Effectors encoded within SPI-2
  7. Secreted effectors encoded outside SPI-2
  8. Conclusions and future directions
  9. Acknowledgements
  10. References

Salmonella enterica uses two functionally distinct type III secretion systems encoded on the pathogenicity islands SPI-1 and SPI-2 to transfer effector proteins into host cells. A major function of the SPI-1 secretion system is to enable bacterial invasion of epithelial cells and the principal role of SPI-2 is to facilitate the replication of intracellular bacteria within membrane-bound Salmonella -containing vacuoles (SCVs). Studies of mutant bacteria defective for SPI-2-dependent secretion have revealed a variety of functions that can be attributed to this secretion system. These include an inhibition of various aspects of endocytic trafficking, an avoidance of NADPH oxidase-dependent killing, the induction of a delayed apoptosis-like host cell death, the control of SCV membrane dynamics, the assembly of a meshwork of F-actin around the SCV, an accumulation of cholesterol around the SCV and interference with the localization of inducible nitric oxide synthase to the SCV. Several effector proteins that are translocated across the vacuolar membrane in a SPI-2-dependent manner have now been identified. These are encoded both within and outside SPI-2. The characteristics of these effectors, and their relationship to the physiological functions listed above, are the subject of this review. The emerging picture is of a multifunctional system, whose activities are explained in part by effectors that control interactions between the SCV and intracellular membrane compartments.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. General features of SPI-2
  5. Physiological activities of the SPI-2 TTSS
  6. Effectors encoded within SPI-2
  7. Secreted effectors encoded outside SPI-2
  8. Conclusions and future directions
  9. Acknowledgements
  10. References

Type III secretion systems (TTSS) are specialized organelles of Gram-negative bacterial pathogens that deliver effector proteins to host cell membranes and cytosol (Hueck, 1998). The TTSS apparatus is a needle-like structure which spans the inner and outer membranes of the bacterial envelope and secretes translocon and effector proteins. The structure of the needle is similar to that of the flagellar basal body, and some of its proteins, including those which form the core of the central channel, are highly conserved between the two systems (Aizawa, 2001). Translocon proteins allow access of effector proteins to the eukarytic cell, probably by forming pores in the host cell membrane and in some cases a connecting channel between the bacterium and the eukaryotic membrane (Frankel et al., 1998). The effector proteins subvert different aspects of host cell physiology and immunity, thereby promoting bacterial virulence.

Salmonella enterica encodes two distinct virulence-associated TTSS within the pathogenicity islands (PAI) SPI-1 and SPI-2. The SPI-1 TTSS of Salmonella enterica serovar Typhimurium ( S. typhimurium ) delivers at least 13 effector proteins through the host cell plasma membrane, most of which are involved in actin cytoskeleton rearrangements, leading to membrane ruffling and Salmonella invasion ( Galan, 1999 ). SPI-1 effectors also induce IL-8 and pathogen-elicited epithelial chemoattractant secretion in intestinal epithelial cells, resulting in transmigration of neutrophils ( Lee et al., 2000 ). The SPI-1 translocon protein SipB binds to and activates caspase-1, leading to the induction of apoptosis in macrophages ( Hersh et al., 1999 ). Mutations which prevent secretion through the SPI-1 TTSS lead to a 10 to 100-fold increase in attenuation in the mouse model of systemic infection, when the bacterial inoculum is administered orally ( Galan and Curtiss, 1989 ; Jones et al., 1994 ; Baumler et al., 1997 ). This lack of complete attenuation of SPI-1 null mutants reflects the ability of Salmonella to disseminate to the liver and spleen from the intestinal tract via an alternative route: carriage within transmigrating, CD-18 expressing phagocytic cells ( Vazquez-Torres et al., 1999 ). After the bacteria reach the spleen and liver, they replicate within membrane-bound compartments, Salmonella -containing vacuoles (SCVs), inside macrophages ( Richter-Dahlfors et al., 1997 ; Salcedo et al., 2001 ). If SPI-1 mutants are administered by the intraperitoneal route, they display no virulence defect, which shows that this TTSS does not have a significant role in bacterial growth within macrophages of the spleen and liver. However, recent evidence indicates that SPI-1 does have an important role in the early stages of SCV biogenesis in epithelial cells ( Steele-Mortimer et al., 2002 ).

The first indication that Salmonella encodes a second TTSS came from a signature-tagged mutagenesis (STM) screen of large numbers of bacterial mutants in the mouse (Hensel et al., 1995). Numerous mutants were obtained that were defective for growth during the systemic phase of infection, and sequencing of regions flanking their transposon insertion sites revealed that a significant proportion encode proteins of a TTSS (Shea et al., 1996). Subsequent mapping of these genes showed that they are clustered on a PAI, which was named SPI-2 (Shea et al., 1996). SPI-2 genes were also identified by sequencing of a region of S. typhimurium genome not present in E. coli (Ochman et al., 1996).

General features of SPI-2

  1. Top of page
  2. Summary
  3. Introduction
  4. General features of SPI-2
  5. Physiological activities of the SPI-2 TTSS
  6. Effectors encoded within SPI-2
  7. Secreted effectors encoded outside SPI-2
  8. Conclusions and future directions
  9. Acknowledgements
  10. References

Although SPI-2 is almost 40 kb in length, genes encoding the secretion system are localized to a region of approximately 26 kb beginning at the centisome 30 end of the island. Here, 31 genes are organized in four operons, termed regulatory, structural I, structural II and effector/chaperone (Shea et al., 1996; Cirillo et al., 1998; Hensel et al., 1998). In addition to several genes encoding evolutionarily conserved structural components of the secreton machinery (Aizawa, 2001) and the translocon proteins SseB, SseC and SseD, there are a few genes that appear to be unique to SPI-2, such as sseE, whose functions are unknown. SseB is similar to EspA of EPEC, and might therefore link the secreton needle to the translocon pore (Beuzón et al., 1999). SseB, SseC and SseD are not required for secretion of effectors, but are necessary for their translocation across the vacuolar membrane (Klein and Jones, 2001; Nikolaus et al., 2001). Hence, these proteins can be operationally defined as translocon components, although their presence in the vacuolar membrane has not been demonstrated directly. In addition to the components of the secreton and translocon, SPI-2 also encodes at least three chaperones: SscA, SscB and SseA. The partners of SscA and SscB have not been defined, but SseA is a chaperone for SseB and SseD (Ruiz-Albert et al., 2003; Zurawski and Stein, 2003). The PAI also encodes a two-component regulatory system which is required for expression of all SPI-2 TTSS genes (Cirillo et al., 1998) as well as several genes located outside SPI-2 which encode effector proteins (Beuzón et al., 2000; Worley et al., 2000; Knodler et al., 2002). In other TTSSs, some translocon proteins have been shown to function as effectors, but there is no evidence yet to indicate whether this might be the case for SseB, SseC or SseD. SpiC, SseF and SseG are proposed to be effector proteins encoded within SPI-2 and are discussed further below.

The SPI-2 TTSS is a multifunctional virulence system (Fig. 1) that is activated following entry of bacteria into eukaryotic cells and facilitates bacterial multiplication in all cell types that have been tested (Ochman et al., 1996; Cirillo et al., 1998; Hensel et al., 1998; Beuzón et al., 2002). Because intracellular growth in cultured cells is correlated with systemic growth in the host (Fields et al., 1986; Leung and Finlay, 1991; Salcedo et al., 2001), this explains the profound attenuation of SPI-2 null mutants in vivo (Hensel et al., 1995; Shea et al., 1996).

image

Figure 1. Intracellular activities of the SPI-2 TTSS. The phenotypes of wild-type and sifA mutant S. typhimurium (green) are shown in relation to different host cell proteins (indicated in box) and vesicular compartments. SCVs do not interact extensively with late endosomes or lysosomes. In macrophages, conflicting evidence implicates the SPI-2 effector protein SpiC (which interacts with the host protein TassC) and the PhoP-Q regulon in this process. SifA, SifB, SseJ, SseF, SseG, PipB and SopD2 are examples of SPI-2 translocated proteins that localize to the SCV and Sifs in epithelial cells. Loss of vacuolar membrane from the sifA mutant requires the action of SseJ.

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Physiological activities of the SPI-2 TTSS

  1. Top of page
  2. Summary
  3. Introduction
  4. General features of SPI-2
  5. Physiological activities of the SPI-2 TTSS
  6. Effectors encoded within SPI-2
  7. Secreted effectors encoded outside SPI-2
  8. Conclusions and future directions
  9. Acknowledgements
  10. References

Through the analysis of SPI-2 null mutants, or individual effectors and their corresponding mutants, SPI-2 has been shown to be required for inhibition of various aspects of endocytic trafficking, including fusion between lysosomes and SCVs (Uchiya et al., 1999), an avoidance of NADPH oxidase-dependent killing by macrophages (Vazquez-Torres et al., 2000; Gallois et al., 2001), delayed apoptosis-like cell death (van der Velden et al., 2000), control of SCV membrane dynamics (Beuzón et al., 2000; Ruiz-Albert et al., 2002), the assembly of a meshwork of F-actin around the SCV (Méresse et al., 2001), cholesterol accumulation around the SCV (Catron et al., 2002) and interference with the localization of inducible nitric oxide synthase (iNOS) to the SCV (Chakravortty et al., 2002). These diverse activities imply the existence of a number of different effector proteins, and indeed several have now been identified, encoded both within and outside of SPI-2 (Table 1).

Table 1. .  SPI-2 effectors and SsrB-regulated proteins.
ProteinFeaturesSsrA-SsrB-dependentTranslocationGene location
  • a

    . ND, not determined.

SpiCInterferes with vesicular traffickingYesSPI-2SPI-2
SseFContributes to Sif formationYesSPI-2SPI-2
SseGContributes to Sif formationYesSPI-2SPI-2
SifARequired for SCV membrane integrityYesSPI-2Pathogenicity islet and Sif formation
SifBTargeted to SifsYesSPI-2Pathogenicity islet
SspH1NDNoSPI-1/SPI-2Gifsy-3 prophage
SspH2Actin remodelling?YesSPI-2Phage
SlrPNDNoSPI-1/SPI-2Plasmid/transposase
SseI/SrfHActin remodelling?YesSPI-2Gifsy-2 prophage
SseJAcyl transferase/SCV membrane dynamicsYesSPI-2Phage
PipBTargeted to SifsYesSPI-2SPI-5
SopD2Targeted to Sifs and late endosomesYesSPI-2Pathogenicity islet
SrfANDYesNDND
SrfBNDYesNDND
SrfCNDYesNDND
SrfDNDYesNDPhage
SrfENDYesNDPhage
SrfGNDYesNDND
SrfINDYesNDPhage
SrfJApoptosis?YesNDND
SrfKNDYesNDPhage
SrfLNDYesNDPhage
SrfMNDYesNDPhage

Effectors encoded within SPI-2

  1. Top of page
  2. Summary
  3. Introduction
  4. General features of SPI-2
  5. Physiological activities of the SPI-2 TTSS
  6. Effectors encoded within SPI-2
  7. Secreted effectors encoded outside SPI-2
  8. Conclusions and future directions
  9. Acknowledgements
  10. References

SpiC

The first SPI-2 effector protein to be identified and characterized was SpiC (Uchiya et al., 1999). Consistent with results from other laboratories (Ishibashi and Arai, 1990; Buchmeier and Heffron, 1991; Rathman et al., 1997), electron microscopic analysis of infected J774 macrophages showed that a significant proportion of vacuoles containing wild-type S. typhimurium do not fuse with endosomes and lysosomes. However, mutation of spiC resulted in a significantly greater proportion of vacuoles undergoing fusion and a strong defect in intramacrophage survival and virulence in mice (Uchiya et al., 1999). spiC encodes a small acidic protein which has no overall similarity to proteins in sequence databases, and lacks the conserved amino-terminal sequence that acts as a secretion/translocation signal in some other effectors (see below). SPI-2-dependent translocation of SpiC into the macrophage cytosol was demonstrated using confocal immunofluorescence microscopy, as well as immunoblot analysis after fractionation of infected cells. SpiC inhibited endosome-endosome fusion in vitro and altered trafficking of the transferrin receptor when expressed from a Sindbis virus vector (Uchiya et al., 1999). More recently, Lee et al. (2002) have identified a novel host cell protein as a target for SpiC, which has been named TassC. TassC was identified by a yeast two-hybrid screen and was also shown to interact with SpiC by pull-down and co-immune precipitation experiments. SpiC appears to exclude TassC from associating with the SCV. The normal cellular location of TassC was not defined, but knock-down of its expression by antisense oligonucleotides promoted the survival of the spiC mutant in macrophages (Lee et al., 2002). If the cellular function of TassC is related to vesicular trafficking and/or fusion, then its interaction with SpiC might provide a mechanistic explanation for the results obtained by Uchiya et al. (1999).

However, work from other groups has shown that SpiC has additional function(s) within the bacterial cell, and has raised questions as to its role as an effector. It has emerged that SpiC is necessary both for in vitro secretion of the SPI-2 translocon proteins SseB, SseC and SseD (Freeman et al., 2002; Yu et al., 2002), and also for translocation of SPI-2 effectors into infected macrophages (Freeman et al., 2002). One conclusion from these findings is that the phenotype of the spiC mutant does not reflect loss of the SpiC protein per se, but is instead likely to be similar, if not identical, to that of a SPI-2 null mutant, as a functional translocon is required for delivery of all effectors to their targets. This explains the observation that a spiC mutant fails to produce Sifs (Guy et al., 2000). Sifs are tubular extensions of the SCV membrane which occur in epithelial cells. They are enriched in lysosomal membrane glycoproteins (lgps) such as LAMP1 (García-del Portillo et al., 1993). Their formation is dependent on another SPI-2 effector, SifA (discussed below). The failure of the spiC mutant to translocate SifA therefore accounts for its inability to form Sifs.

Of course it is possible that SpiC could have dual function, acting both as effector and in promoting translocon protein secretion in the bacterial cell, but other data are more difficult to reconcile. First, under in vitro bacterial growth conditions that allow secretion of other SPI-2 translocon and effector proteins, the groups of Miller, Hensel and Holden have been unable to detect secretion of epitope-tagged or untagged SpiC (Freeman et al., 2002; Hansen-Wester et al., 2002a; Yu et al., 2002). Second, Freeman et al. (2002) were unable to detect translocation of epitope-tagged SpiC into infected macrophages by immunofluorescence microscopy. There may be relatively trivial explanations for these inconsistencies. For example, the environmental signal triggering SpiC secretion might be different from that of other secreted proteins examined, and might not be present in the in vitro growth medium used. As there was no control to show that the epitope-tagged SpiC was expressed to detectable levels in bacterial cells in the immunofluorescence analysis by Freeman et al. (2002), it is difficult to be certain that SpiC was not translocated under their conditions. However, it is clear that there are a number of issues to be resolved and more work is required to clarify the potentially complex functions of SpiC.

SseF and SseG

The SPI-2-encoded proteins SseF and SseG have no similarity to sequences in the databases but are similar to each other and both contain predicted transmembrane helices (Cirillo et al., 1998; Hensel et al., 1998). Strains carrying mutations in sseF or sseG exhibit only slight defects in replication in macrophages and virulence in mice (Hensel et al., 1998), and are not defective for translocation of other effectors (Kuhle and Hensel, 2002). SPI-2-dependent secretion of M45 epitope-tagged SseF and SseG has been demonstrated in vitro by Hansen-Wester et al. (2002a). In the S. typhimurium SL1344 genetic background, sseF and sseG mutants were found to be severely defective in Sif production in epithelial cells (Guy et al., 2000), but in the 12023 (14028s) strain, these mutations still allowed the formation of poorly developed, morphologically abnormal Sifs (Kuhle and Hensel, 2002). As Sifs are extensions of the SCV membrane, this might suggest a role for these proteins in vacuolar membrane dynamics. Immunofluorescence microscopy of M45-tagged SseF and SseG showed that both proteins are translocated to the SCV membrane, Sifs and other endosomal compartments (Kuhle and Hensel, 2002).

Secreted effectors encoded outside SPI-2

  1. Top of page
  2. Summary
  3. Introduction
  4. General features of SPI-2
  5. Physiological activities of the SPI-2 TTSS
  6. Effectors encoded within SPI-2
  7. Secreted effectors encoded outside SPI-2
  8. Conclusions and future directions
  9. Acknowledgements
  10. References

Several effectors contain a conserved N-terminal translocation motif

DNA sequence analysis of the chromosomal region surrounding the PhoP-activated gene pagJ led to the identification of a gene encoding a 700-amino-acid protein that was designated SspH1 (Miao et al., 1999). Probes derived from sspH1 detected a second hybridizing sequence that contained a gene, named sspH2, which encodes a protein of similar size and 69% identity to SspH1. The SspH1 and SspH2 proteins contain leucine-rich repeats. SspH1 was shown to be a target of the SPI-1 and SPI-2 TTSSs, whereas SspH2 translocation was restricted to the SPI-2 TTSS. Both proteins are similar to the Shigella flexneri and Yersinia TTSS effectors IpaH and YopM. A third protein that contains leucine-rich repeats and is similar to SspH1 and SspH2 is SlrP, which was identified as contributing to virulence in mice by an STM screen for genes involved in host adaptation to mice and calves (Tsolis et al., 1999). In contrast to SlrP, SspH1 and SspH2 are not involved in murine systemic infection but do contribute to the virulence of S. typhimurium in calves (Miao et al., 1999).

In a later report by Miao and Miller (2000), a further four ORFs which encode likely effector proteins were identified on the basis of the similarity of their amino termini to those of SspH1, SspH2 and SlrP. These were SifA (Stein et al., 1996) and the previously undescribed proteins SseI, SseJ and SifB. The sequence of SseI gives no clue to its possible function. SseJ is 30% identical to the glycerophospholipid:cholesterol acyltransferase of Aeromonas and includes three residues that make up the catalytic triad of the enzyme, within three conserved regions that exist in all members of this family of acyl transferases. SifB shares 30% overall amino acid identity with SifA, but SifA has no significant similarity to other database entries.

Use of transcriptional reporter fusions showed that expression of sspH2, sseI, sseJ, sifA and sifB is dependent on the SPI-2 TTSS regulator SsrA-SsrB. In contrast, sspH1 and slrP expression was independent of both SsrA-SsrB and the SPI-1 regulator HilA, suggesting that these two genes might be expressed constitutively (Miao and Miller, 2000). To determine if the amino-terminal conserved region of these proteins is required for their translocation, fusions were made to the Bordetella pertussis cyaA gene. Protein fusions to catalytic domains of adenylate cyclase toxin catalyse the conversion of ATP to cAMP only in the presence of calmodulin in the cytoplasm of eukaryotic cells. Hence, the cyaA gene acts as a very useful and sensitive translocation reporter (Sory and Cornelis, 1994). Increased cAMP levels inside host cells, caused by the translocation of SspH1, SspH2, SlrP, SseI and SseJ fusions across the phagosome membrane, was shown to be dependent upon the SPI-2 TTSS. SspH1 and SlrP fusions were also translocated across the plasma membrane by the SPI-1 TTSS. An alignment of the putative translocation signals of all seven proteins revealed a conserved region (WEKI(I/M)XXFF) present in all proteins at amino acid residues 34–41 of the alignment. An SspH1-CyaA fusion carrying a deletion in this motif eliminated its translocation by both SPI-1 and SPI-2 TTSSs (Miao and Miller, 2000). SPI-2-dependent translocation of SifB to the SCV membrane and Sifs has been demonstrated by immunofluorescence microscopy of an epitope-tagged protein (Freeman et al., 2003). Inactivation of sseI or sifB did not result in a detectable virulence defect in systemic infection of mice (Ruiz-Albert et al., 2002), although the high inoculum and relatively short duration of infection may have masked subtle defects. The lack of a virulence defect for sseI and sifB mutants has been confirmed in separate studies (Ho et al., 2002; Freeman et al., 2003). Both sifA and sseJ contribute to systemic infection and their functions are discussed further below.

More recently, another SPI-2 effector (SopD2) has been identified that is 42% identical in amino acid sequence to SopD, a SPI-1 effector that mediates inflammation and fluid secretion in bovine ileal mucosa (Jones et al., 1998). An S. typhimurium sopD2 mutant has a virulence defect in mice, and the protein was shown to be translocated via the SPI-2 TTSS in epithelial cells to the SCV membrane, Sifs and late endocytic compartments (Brumell et al., 2003). Interestingly, the N-terminal 150 amino acids of SopD2 fused to GFP was sufficient to target the protein to late endocytic compartments and to induce their swelling. This was an important finding because it shows that the N-terminal region of SopD2 is likely to contribute to effector function as well being required for secretion/translocation (Brumell et al., 2003).

sspH1 , sspH2 and sseI are all located within lysogenic bacteriophages ( Miao and Miller, 2000 ; Figueroa-Bossi et al., 2001 ; Hansen-Wester et al., 2002b ). This is also true for at least one SPI-1 effector ( Mirold et al., 2001 ), and may represent a common mechanism for the dissemination of Salmonella effectors. sspH2 is encoded within an uncharacterized bacteriophage whereas sseI and sspH1 are within the Gifsy-2 and Gifsy-3 prophages respectively ( Figueroa-Bossi et al., 2001 ). These prophages are competent for lytic growth and lysogeny of naive Salmonella ( Figueroa-Bossi and Bossi, 1999 ). This suggests that sseI and sspH1 are actively disseminating throughout the salmonellae. In accordance with this, sspH1 and sseI are present in a minority of Salmonella serotypes, whereas sspH2 is more widely distributed ( Miao et al., 1999 ; Tsolis et al., 1999 ; Hansen-Wester et al., 2002b ). Indeed, the seven non-SPI-2 genes encoding translocated effectors have a variable G + C content, ranging from 38% for sifB to 55% for sspH2 , implying that they have been acquired by horizontal transmission at different times or from different sources. sopD2 and sseJ are pseudogenes in Salmonella typhi ( Parkhill et al., 2001 ), suggesting that their functions might be host-specific.

SifA

The extensive reorganization of host cell lgps into Sifs and SCV membranes in infected epithelial cells was first observed by García-del Portillo et al. (1993). Subsequently, a genetic screen carried out by the same laboratory resulted in the identification of a gene, designated sifA, that is required for Sif formation (Stein et al., 1996). The location of sifA in the potBCD operon, its low G + C content, and the presence of direct repeats flanking the gene all suggest that sifA was acquired as a result of a horizontal transfer. sifA was shown to be necessary for Salmonella virulence, although curiously the mutant grew better than the wild-type strain in epithelial cells (Stein et al., 1996). The discovery that SifA is an important SPI-2 effector has emerged from work of several laboratories. Miao and Miller (2000) found that SifA is regulated by the SPI-2 SsrA-SsrB two component system and that it contains the conserved N-terminal amino acid sequence discussed above. Further evidence for a functional link between SifA and SPI-2 came from: (i) the finding that SPI-2 null mutants fail to make Sifs (Beuzón et al., 2000; Guy et al., 2000; Brumell et al., 2001a); (ii) a genetic test indicating that the two loci interact functionally in vivo (Beuzón et al., 2000); (iii) the finding that sifA expression occurs following bacterial entry into host cells in an ssrA-dependent manner (Beuzón et al., 2000) and (iv) that sifA mutants have a replication defect in macrophages (Beuzón et al., 2000; Brumell et al., 2001a). The replication defect occurs because the sifA mutant loses its vacuolar membrane (Beuzón et al., 2000). In macrophages, this exposes bacteria to a cytosolic killing activity (Beuzón et al., 2002) which might be ubiquicidin, a small cationic protein derived from the ribosomal protein S30 (Hiemstra et al., 1999). Interestingly, although the vacuolar membrane enclosing sifA mutants is also lost in epithelial cells, the killing activity is not present, providing an explanation for the observation that mutant bacteria grow at a greater rate than wild-type bacteria (Beuzón et al., 2002; Brumell et al., 2002). The sifA mutant phenotype can be complemented if SifA is expressed ectopically in host cells infected with mutant bacteria (Beuzón et al., 2000). SPI-2 TTSS-dependent translocation of a haemagglutinin (HA)-tagged SifA to the SCV membrane and Sifs was subsequently shown by fractionation experiments and immunofluorescence microscopy (Brumell et al., 2002). SifA function is dependent on the six C-terminal amino acids of the protein (CLCCFL). This peptide is required to anchor SifA to membranes and is a probable site for lipid modification (Boucrot et al., 2003). Expression of SifA in HeLa cells results in vacuolation of LAMP1-positive compartments and formation of structures resembling Sifs (Brumell et al., 2001b), suggesting that SifA is largely sufficient for Sif formation.

What is the function of SifA? This effector seems to be required both for recruitment of LAMP1-containing vesicles to the bacterial microcolony (Ruiz-Albert et al., 2002), and fusion of these with the SCV membrane, or its stabilisation (Beuzón et al., 2000). These activities could ensure a progressive increase of SCV membrane surface area to accommodate replicating bacterial cells. However, it should also be borne in mind that despite the requirement for a net gain of vacuolar membrane during bacterial replication, bacterial replication per se does not play a significant role in loss of vacuolar membrane of sifA mutant bacteria (Ruiz-Albert et al., 2002). At the biochemical level, SifA might stimulate or inhibit a protein that controls vesicle targeting and membrane fusion in the cell. Clearly, identification of the cellular target(s) of SifA is the essential next step towards an understanding of its function.

SseJ

Mutation of sseJ results in a modest attenuation of virulence during systemic infection of mice, which is correlated with a small replication defect in primary macrophages (Ruiz-Albert et al., 2002; Freeman et al., 2003). Like SifA, expression of SseJ in Hela cells produces a dramatic reorganization of LAMP1-positive vesicles. However, in the case of SseJ, these take the form of globular membranous compartments (GMCs) which are composed of material that is characteristic of the SCV membrane and Sifs, enclosed by SseJ. SseJ also localizes to the vacuolar membrane and Sifs in infected, transfected cells (Ruiz-Albert et al., 2002), and when expressed from bacteria in infected cells (Kuhle and Hensel, 2002; Freeman et al., 2003). When SseJ and SifA are co-expressed in HeLa cells, GMC-like structures are produced, with SifA enclosed by SseJ. Although these structures (and GMCs) are unlikely to have physiological relevance, they suggested a functional link between SseJ and SifA (Ruiz-Albert et al., 2002). Of particular interest was the observation that a sifA sseJ double mutant does not lose its vacuolar membrane, implying that loss of the vacuolar membrane around a sifA mutant requires the action of SseJ (Ruiz-Albert et al., 2002). Because SseJ is predicted to be an acyl transferase, one possiblity is that its function is to modify SCV membrane lipids to facilitate budding and/or scission. In the absence of SifA, SseJ activity on the SCV membrane could result in its destabilisation and eventual loss.

Other members of the SsrA-SsrB regulon

Using a genetic screen of 20 000 mutants, Worley et al. (2000) identified numerous genes, located outside SPI-2, that are regulated by SsrB. Ten genes were characterized further, all of which appear to have been acquired by lateral transfer. Their level of ssrB-dependent activation in macrophages ranged from two- to 80-fold. The most highly regulated gene, srfH, has similarity to sspH1, sspH2 and slrP, and is located in a Gifsy-2 prophage. It was recently confirmed by Figueroa-Bossi et al., 2001) that srfH and sseI are the same gene. The product of another gene, named srfJ, shares striking amino acid similarity with the human lysosomal glucosyl ceramidase. In several systems ceramide initiates the stress-activated protein kinase (SAPK)/c-jun kinase (JNK) cascade to signal apoptosis. Therefore SrfJ might be the effector responsible for SPI-2-dependent apoptosis (van der Velden et al., 2000), but this has yet to be examined. Mutation of srfJ leads to a very mild attenuation of virulence in mice (Ruiz-Albert et al., 2002). The possible functions of most of these genes remain unknown, and indeed it has yet to be demonstrated that they are SPI-2 effectors.

Further organizational complexity of genes encoding Salmonella effectors has been revealed by the discovery that in another PAI, SPI-5, two adjacent genes encoding the SPI-1 TTSS effector SigD/SopB and a novel SPI-2 effector, PipB, are co-ordinately regulated by their respective regulatory systems (Knodler et al., 2002). An HA-tagged PipB was shown to be translocated to the SCV membrane and Sifs (Knodler et al., 2002). However, in contrast to SifA, PipB was not required for the formation or maintenance of either the Sifs or the SCV membrane. No defect in intracellular survival or virulence could be detected for a pipB mutant, perhaps reflecting functional redundancy with another effector. PipB translocation signals were localized to its N-terminal domain. However, this region does not contain the WEKI(I/M)XXFF motif present in the translocation domain of some of the other SPI-2 secreted effectors. How PipB, SpiC, SseF and SseG are recognized and translocated by the SPI-2 TTSS is unknown. It is possible that all effectors have N-terminal three dimensional similarity not apparent from primary sequence; alternatively, different mechanisms might be employed to target effector protein secretion and translocation.

Further work is required to establish if the genes described above encode the complete repertoire of SPI-2 effectors. It would be beneficial if bacterial growth conditions could be found which induce secretion of substantial amounts of SPI-2 effectors in vitro, as this would enable their identification by polyacrylamide gel electrophoresis followed by microsequencing or mass spectrometry. However, even when large culture volumes are used in conjunction with optimized secretion conditions, we have only been able to detect low amounts of secreted SPI-2 effectors by Coomassie blue or silver staining of gels (J. Garmendia and D. W. Holden, unpubl. data).

Other SPI-2 effector functions

In addition to influencing vesicular trafficking and SCV membrane dynamics, SPI-2 has been implicated in other intracellular activities. These studies have generally involved the use of SPI-2 null mutant strains, and the effectors responsible have yet to be identified.

Avoidance of the oxidative burst

One of these activities is the ability of the SCV to avoid the damaging effects of the respiratory burst in macrophages. This came from the finding that the virulence of SPI-2 null mutants is restored to a considerable degree in gp91phox knock-out animals, which lack NADPH oxidase activity (Vazquez-Torres et al., 2000). Components of the NADPH oxidase complex, including p22 and p47 subunits and cytochrome b558 are excluded from the SCV in a SPI-2 dependent manner (Vazquez-Torres et al., 2000; Gallois et al., 2001). The SPI-2 effector(s) responsible for this exclusion could act through tumor necrosis factor receptor p55, as recruitment of the NADPH oxidase to vacuoles containing SPI-2 mutants has been shown to require this receptor (Vazquez-Torres et al., 2001).

Cytotoxicity

Salmonella typhimurium can induce a rapid programmed cell death in macrophages via the SPI-1 TTSS through the action of the secreted protein SipB ( Chen et al., 1996 ; Monack et al., 1996 ; Hersh et al., 1999 ). However, S. typhimurium was also found to induce a delayed apoptosis in infected macrophages beginning 12–13 h after ingestion, in the absence of a SPI-1 secretion system or SipB ( van der Velden et al., 2000 ). This process was dependent on a functional SPI-2 TTSS, but was not simply a consequence of intracellular bacterial proliferation because a mutant with an unrelated replication defect was as cytotoxic as the wild-type strain ( van der Velden et al., 2000 ). Recent reports suggests that the apoptotic-like process is in fact an unusual form of necrosis ( Brennan and Cookson, 2000 ; Watson et al., 2000 ). In vivo , this SPI-2-dependent cytotoxicity could facilitate bacterial spread within the spleen and liver. In agreement with this, dying cells containing Salmonella have been observed in livers of infected mice ( Richter-Dahlfors et al., 1997 ).

Although caspase-1 has been shown to be necessary for orally administered Salmonella to colonize murine Peyer's patches, inoculation of caspase-1–/– mice with S. typhimurium via the intraperitoneal route resulted in a level of bacterial colonization of the spleen that was no different from that found in wild-type mice (Monack et al., 2000). Experiments with bone marrow-derived macrophages showed that SPI-2-induced macrophage cytotoxicity is only partly dependent on caspase-1 (Monack et al., 2001). These results suggest either that SPI-2-dependent cytotoxicity has no significant role in promoting bacterial proliferation in the spleen in vivo, or that it does have, but by a mechanism that is independent of caspase-1. The SPI-2 secreted effector(s) responsible for this macrophage cytotoxicity have not been identified. In Salmonella dublin, the SPI-2 locus has been shown to be required for apoptosis of intestinal epithelial cells (Paesold et al., 2002).

Intracellular actin assembly

Several hours after uptake by a variety of host cells, the SPI-2 TTSS induces the assembly of F-actin in close proximity to the SCV membrane and Sifs (Méresse et al., 2001; Brumell et al., 2002; Miao et al., 2003). The G-actin sequestering agent latrunculin B prevented the accumulation of actin in Salmonella-infected cells and prevented bacterial replication in macrophages. In addition, latrunculin B treatment caused vacuolar membrane loss from a significant proportion of wild-type bacteria (Méresse et al., 2001). Therefore the physiological role of the SPI-2 dependent actin meshwork would appear to be connected with vacuolar membrane dynamics. It might stabilize the structure of the membrane, or facilitate transport of vesicles destined to fuse with the SCV. The effector(s) controlling this process could be unknown, as strains carrying mutations in either sifA, sifB, slrP, srfJ, sseE, sseF, sseG, sseI, sspH2, sseI sspH2 or sseJ were all proficient for actin assembly (Méresse et al., 2001; Yu et al., 2002; Miao et al., 2003). However, in common with actin rearrangements induced by TTSSs of other bacterial pathogens, the process is likely to be modulated by the concerted actions of several effectors. Indeed, it has recently been shown that SspH2 and SseI co-localize with the SPI-2 actin meshwork and a two-hybrid screen revealed that they interact with the actin crosslinking protein filamin through their conserved amino-terminal domains (Miao et al., 2003). SspH2 was also shown to bind the actin-binding protein profilin. In addition, another S. typhimurium virulence protein, SpvB, which has been shown to ADP-ribosylate actin (Lesnick et al., 2001; Tezcan-Merdol et al., 2001), strongly inhibited SPI-2 associated actin polymerization. However, the physiological relevance of this result is not clear, as genetic studies have showed that SPI-2 and the spv operon function independently in vivo (Shea et al., 1999).

INOS localization

Yet another characteristic of SCVs that appears to be SPI-2-dependent is alteration of iNOS localization in infected macrophages (Chakravortty et al., 2002). Confocal microscopic examination of infected RAW 264.7 and peritoneal macrophages revealed that the subcellular localization of iNOS was dramatically different in macrophages infected with wild-type Salmonella or SPI-2 mutants. iNOS was shown to co-localize strongly with SPI-2 mutants but not with the wild-type strain. It was suggested that increased killing of SPI-2 mutants by nitrogen-derived radicals contribute to their reduced intracellular proliferation. No difference was observed between wild-type Salmonella- and SPI-2 mutant-infected cells in terms of iNOS protein levels or nitrite production, indicating that SPI-2 is not involved in inhibiting this activity. Curiously, when the growth of SPI-2 mutant and wild-type bacteria was examined by mixed infections in spleens and livers of iNOS–/– mice, the SPI-2 mutant was strongly out-competed up to three days post inoculation, but then grew faster than the wild-type strain to reach equivalent numbers in the liver 48 h later. The reason for this is not clear, but it clearly warrants further investigation.

Cholesterol accumulation near the SCV

Recent studies have found that cholesterol is recruited to SCVs in epithelial cells and macrophages (Brumell et al., 2001b; Catron et al., 2002). In macrophages, recruitment of cholesterol and the cholesterol-associated glycosylphosphatidylinositol (GPI)-anchored protein CD55 were shown to be dependent on a functional SPI-2 TTSS (Catron et al., 2002), suggesting the involvement of a SPI-2 effector. However, other mutants with intracellular replication defects were not analysed for cholesterol recruitment, so it is conceivable that the phenotype is not a specific function of SPI-2 but a more general characteristic of replicating bacteria. In any event, the recruitment of cholesterol is striking and is consistent with the emerging theme linking SPI-2 and membrane dynamics. It is interesting to note that GMCs induced by expression of SseJ also contain cholesterol (Ruiz-Albert et al., 2002).

Conclusions and future directions

  1. Top of page
  2. Summary
  3. Introduction
  4. General features of SPI-2
  5. Physiological activities of the SPI-2 TTSS
  6. Effectors encoded within SPI-2
  7. Secreted effectors encoded outside SPI-2
  8. Conclusions and future directions
  9. Acknowledgements
  10. References

An obvious conclusion from the studies described above is that the functions of the SPI-2 secretion system are complex, and involve the translocation of numerous effectors into and possibly across the vacuole membrane. However, although several translocated proteins have now been identified, effector function has been ascribed to only a few. It seems clear that the functions of SifA, SseJ and SPI-2-dependent actin remodelling all relate to SCV membrane dynamics. Regulating the acquisition, fusion and fission of vacuolar membrane must be of fundamental importance for intravacuolar pathogens such as Salmonella, and it would not be surprising if more effectors were found to be involved in this process. Some controversy exists regarding the function(s) of SpiC, and its proposed role in inhibiting different aspects of cellular trafficking needs further investigation. The effectors that are responsible for SPI-2-dependent cytotoxicity, cholesterol accumulation, avoidance of iNOS and the respiratory burst complex, and intracellular actin assembly, all need to be identified and their specific biochemical functions investigated. Of course it is quite possible that some of these phenotypes reflect different consequences of action by the same effector protein. For example, if SifA mediates fusion of a specific class of membrane vesicles with the SCV, then the altered composition of the resulting vacuolar membrane might account for other SPI-2 phenotype(s).

A major challenge in the future will be to determine the functions of the translocated proteins with no obvious mutant phenotype, such as SspH2, SifB and SseI. This may prove difficult to achieve if there is functional redundancy among SPI-2 effectors. If so, phenotypic analysis of strains carrying mutations in multiple effector genes will be necessary. We need to know if these proteins represent the complete repertoire of SPI-2 effectors or if others exist. Other important questions concern the specific signal(s) in effectors that allow SPI-2 mediated secretion and translocation, and the kinetics of their translocation. Are they translocated simultaneously or sequentially? More detailed studies are required but the latter is a possibility given that translocated SpiC was detected by immunofluorescence as early as 1 h after bacterial uptake in macrophages (Uchiya et al., 1999), whereas translocation of epitope-tagged SseF and SseG could not be detected until 3 h later (Kuhle and Hensel, 2002), and in HeLa cells translocated epitope-tagged PipB could not be detected by immunofluorescence before 6 h after infection (Knodler et al., 2002). It is appealing to consider that ordered translocation kinetics might correspond to different requirements for SCV biogenesis and bacterial multiplication over time.

Over the last decade, detailed studies into the functions of the effector proteins translocated by the TTSS of Escherichia coli, Shigella, Salmonella SPI-1, Yersinia and Pseudomonas have provided major insights into the molecular mechanisms underlying the ability of these pathogenic bacteria to attach to host cells, invade them and avoid phagocytosis. Now that several SPI-2 effectors and a variety of physiological functions for SPI-2 have been identified, we can expect that continued investigations into this TTSS will also yield important information on how Salmonella is able to survive and replicate within its vacuolar compartment inside host cells.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. General features of SPI-2
  5. Physiological activities of the SPI-2 TTSS
  6. Effectors encoded within SPI-2
  7. Secreted effectors encoded outside SPI-2
  8. Conclusions and future directions
  9. Acknowledgements
  10. References

We thank Dr Kate Unsworth for valuable comments on the manuscript. Work in D.W. Holden's laboratory is supported by grants from the MRC and Wellcome Trust (UK).

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  2. Summary
  3. Introduction
  4. General features of SPI-2
  5. Physiological activities of the SPI-2 TTSS
  6. Effectors encoded within SPI-2
  7. Secreted effectors encoded outside SPI-2
  8. Conclusions and future directions
  9. Acknowledgements
  10. References
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