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Characterization of effector proteins translocated via the SPI1 type III secretion system of Salmonella typhimurium
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Int. J. Med. Microbiol. 291, 479-485 (2002) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ijmm
Characterization of effector proteins translocated via the SPI1 type III secretion system of Salmonella typhimurium Kristin Ehrbar, Susanne Mirold, Andrea Friebel, Silke Stender, Wolf-Dietrich Hardt Max von Pettenkofer-Institut, Pettenkoferstraße 9a, D-80336 München, Germany
Abstract Salmonella spp. employ a conserved type III secretion system encoded within the pathogenicity island 1 (SPI1; centisome 63) to translocate effector proteins into the host cytosol. The translocated effector proteins trigger diverse responses including bacterial internalization. In a mutation analysis we have defined the set of effector proteins mediating tissue culture cell invasion. This set includes sopE2 (centisome 40–42), sopB (SPI5, centisome 20) and in the case of S. typhimurium SL1344 also the phage-encoded effector sopE (SopEΦ, centisome 59–60). A triple mutant SL1344 derivative deficient of SopE, SopE2 and SopB was more than 100-fold attenuated in tissue culture cell invasion. Phylogenetic analyses indicate that the last common ancestor of all contemporary Salmonella lineages already harbored all genes necessary for host cell invasion, namely the SPI1 type III secretion system, sopE2 and sopB. SopE, which is 70 % identical to sopE2 is only present in some Salmonella strains and emerged later well after the divergence of the contemporary Salmonella lineages. Interestingly, S. typhimurium strains that harbor sopE are associated with epidemics, arguing that sopE is one of the factors determining the “fitness” of a strain. We found that SopE can specifically activate a different set of host cellular RhoGTPases than SopE2. This allows the bacteria to fine tune host cellular responses very precisely and may offer an explanation for the improved epidemic fitness of sopE-positive S. typhimurium strains. Key words: Salmonella – type III secretion – evolution – phage – Cdc42
Introduction Infections with Salmonella spp. are among the leading causes of bacterial enteritis in developed countries. The type of disease can range from a mild gastroenteritis to systemic infection and is determined by the virulence characteristics of the Salmonella strain as well as by the host species. Detailed phylogenetic analysis has revealed that Salmonella spp. have diverged from the Escherichia spp. about 100–160 million years ago (Ochman and Wilson, 1987). S. enterica harbors about 400–800 kb of DNA including at least 5 separate ‘pathogenicity islands’ necessary for disease progression which are absent from the E. coli genome (reviewed by
Groisman and Ochman (1996)). It is thought that much of the additional DNA acquired via horizontal gene transfer has played a role in the evolution of Salmonella spp. as pathogens (Ochman and Moran, 2001). Two of these additional DNA segments termed Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) encode type III secretion systems (Hueck, 1998). Type III secretion systems have been identified in many gram-negative bacteria living in close association with animals or plants (Galán and Collmer, 1999). Sequence similarities and evidence from electron microscopy studies suggest that type III secretion systems have evolved from bacterial flagella systems. They allow to inject (translocate) bacterial (effector) proteins directly
Corresponding author: Wolf-Dietrich Hardt, Max von Pettenkofer-Institut, Pettenkoferstraße 9a, D-80336 München, Germany, Phone: +49 89 5160 5263, Fax: +49 89 5160 5223, E-mail: [email protected] 1438-4221/01/291/6-7-479 $ 15.00/0
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into the cytosol of host cells to manipulate host cellular signalling cascades. Acquisition of SPI1 has been a key event in the early phase of the evolution of Salmonella as a pathogen (Groisman and Ochman, 1996) and the genes for the SPI1 type III secretion apparatus are present in all contemporary Salmonella lineages (Boyd et al., 1997; Li et al., 1995). Work on strains of S. enterica subspecies 1 serotypes Typhimurium and Dublin has shown that the SPI1 type III secretion system mediates penetration of the host ileal mucosa and the induction of PMN transmigration and diarrhea in the bovine ileum (reviewed by Wallis and Galyov (2000)). In tissue culture cells proteins translocated by the SPI1 type III secretion system induce cell death in macrophages, chloride secretion, IL8 production, membrane ruffling and bacterial entry into non-phagocytic cell lines (reviewed by Galán and Zhou (2000)). So far, 12 different translocated effector proteins are known to be translocated into host cells via the SPI1 type III secretion system (Tab. 1). Studying the function of each of these effector proteins in Salmonella virulence has proven to be quite difficult, since disruption of genes for a single translocated effector protein has often resulted only in minor virulence deTable 1. Effector proteins translocated via the SPI1 type III secretion system. Effector
Location
(Proposed) molecular function
Reference
SipA
SPI1
F-actin stabilization and bundling enhances SipC-mediated actin polymerization?
Zhou et al., 1999a,b McGhie et al., 2001
SipB#
SPI1
cell death in macrophages
Hersh et al., 1999
SipC#
SPI1
actin polymerization/ bundling
McGhie et al., 2001 Hayward and Koronakis, 1999
SptP
SPI1
GAP for RhoGTPases
Fu and Galan, 1999
AvrA
SPI1
?
Hardt et al., 1997
SopA
cs 41-42
?
Wood et al., 2000
SopB
SPI5, cs 20
PI phosphatase
Galyov et al. 1997
SopE
SopEF cs 60
GEF for RhoGTPases
Hardt et al., 1998b Wood et al., 1996
SopE2
cs 40-42
GEF for RhoGTPases
Stender et al., 2000
SopD
cs 64
?
Jones et al., 1998
SspH1*
GIFSY-3 cs 23-25
?
Miao et al., 1999 Figueroa-Bossi et al., 2001
SlrP*
cs 18-19
?
Tsolis et al., 1999
* also translocated via the SPI2 type III secretion system. # also involved in translocation of the other effector proteins (Collazo and Galan, 1997).
fects (see Table 1). Recent work using Typhimurium mutants lacking multiple effector proteins suggests that this might be due to functional redundancy between different translocated effector proteins (Jones et al., 1998; Stender et al., 2000; Zhou et al., 2001; Mirold et al., 2001). The identification and characterization of the translocated effector proteins have also been complicated by the fact that many key effector proteins are not encoded within SPI1 but in distant regions of the chromosome (Fig. 1). In this article we will review the properties of Salmonella effector proteins translocated via the SPI1 type III secretion system with a focus on those effector proteins mediating host cell invasion.
Identification of the effector proteins mediating host cell invasion Soon after the discovery of the first genes encoding functional components of the SPI1 type III secretion apparatus it was found that it is involved not only in the early gut-associated stages of the murine Typhimurium infection but also in the triggering of host cytoskeletal rearrangements and tissue culture cell invasion (reviewed by Galán (1999)). The translocated effector proteins triggering these responses have been identified by subsequent genetic analyses and by sequencing of proteins that were secreted by wild-type Salmonella strains but not by mutants with a disrupted type III secretion apparatus (Hueck et al., 1995; Kaniga et al., 1995a and b; Wood et al., 1996). However, considerable efforts were still required to unequivocally identi-
Fig. 1. Chromosomal map of S. enterica subspecies I serovar Typhimurium. The locations of SPI1 and of the known effector protein genes within and outside SPI1 are shown.
S. typhimurium SPI1 type III effector proteins
fy those effector proteins which modulate host cellular signalling necessary for invasion. Typhimurium strains carrying single mutations in genes encoding secreted proteins could be grouped into three categories: 1. Secreted proteins which are part of the secretion apparatus and absolutely required for the secretion of all other secreted proteins. SpaO and invJ mutants are non-invasive (Collazo and Galán, 1996). 2. Secreted proteins required for delivery of proteins of the 3rd group into the host cell. SipB, sipC and sipD mutants still secrete the other proteins into the culture media, but they are non-invasive (Collazo and Galán, 1997). However, it should be pointed out that SipB and SipC might serve dual functions acting as “translocases” but also as effector proteins once they arrive in the host cytosol. 3. Genuine effector proteins that are not required for secretion or translocation of other proteins. This includes SipA, SptP, AvrA, SopB, SopD, SopE, SopE2 and probably also SopA, SlrP and SspH1. Mutation analyses revealed that only sopB, sopE and sopE2 mutants were mildly (about 2-fold) attenuated in tissue culture cell invasion (Wood et al., 1996; Hardt et al., 1998a; Hong and Miller, 1998; Wood et al., 1998; Bakshi et al., 2000; Stender et al., 2000). These defects were much weaker than the defects (500-fold attenuation) observed with Salmonella mutants harboring an inactivated SPI1 type III translocation apparatus. Biochemical analyses demonstrated that SopE and SopE2 act as G-nucleotide exchange factors for host cellular RhoGTPases like Cdc42 (Hardt et al., 1998a; Stender et al., 2000). RhoGTPases are key regulators of diverse cellular activities including the expression of pro-inflammatory cytokines and the structural organization of the eukaryotic actin cytoskeleton. Activation of Cdc42 and Rac1 is known to lead to membrane ruffling (reviewed by Bishop and Hall (2000)). SopB
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harbors two consensus motifs for eukaryotic inositol polyphosphate 4-phosphatases and alters PI phosphate levels inside infected host cells (Norris et al., 1998; Zhou et al., 2001; Feng et al., 2001). Transfection experiments with SopE, SopE2 or SopB expression vectors demonstrated that each of these effector proteins is sufficient to induce dramatic cytoskeletal rearrangements in tissue culture cells or mediate bacterial internalization (Hardt et al., 1998a; Stender et al., 2000; Zhou et al., 2001). This evidence suggested that SopE, SopE2 and SopB have at least partially redundant functions and that they cooperate to mediate efficient host cell invasion. Indeed, double mutants of Typhimurium SL1344 lacking two of the three effector proteins are 4–10-fold less invasive than the isogenic wild-type strain, and a triple mutant with disrupted genes for SopE, SopE2 and SopB is virtually unable to enter host cells (Tab. 2). This defect could be complemented with SopE or SopE2 expression vectors and to a significant extent also with a SopB expression vector. Together, these observations argue that SopE, SopE2 and SopB jointly mediate host cell invasion. Other translocated effector proteins might also contribute to invasion. Indeed, a sipA mutant is somewhat less invasive and SipA can directly bind to actin, stabilizes F-actin and modulates the bundling of F-actin filaments (Zhou et al., 1999a, b). However, induction of cytoskeletal rearrangements by SipA alone has not been demonstrated. In vitro experiments show that purified (refolded) SipC can mediate de novo actin polymerisation, and microinjection of purified SipC leads to dramatic rearrangements in the actin distribution in tissue culture cells. In addition it has been found that SipA cooperates with SipC in remodelling actin structures in semi-permeabilized cells (McGhie et al., 2001). Taken together, these observations argue that some effector function of SipC and/or SipA might ac-
Table 2. Several effector proteins cooperate to mediate host cell invasion. Strain
Relevant phenotype
Invasiveness (%)1
Macrophage cytotoxicity2 (%)
Reference
SL1344 SB856 M202 M516 M511 M516 + pM149 M516 + pSB1130 M516 + pM515 SB161
wild-type sopE sopE, sopE2 sopE, sopE2, sopB sopE, sopE2, sopB, sopD sopE, sopE2, sopB, (psopE2) sopE, sopE2, sopB, (psopE) sopE, sopE2, sopB, (psopB) invG
100 53.3 29 28.4 3.9 .95 1.0 .93 0.4 80.8 31 213.0 81 12.2 1.2 .17 0.1
100 94 86 90 85 87 90 17 3
Hoiseth and Stocker, 1981 Hardt et al., 1998a Stender et al., 2000 Mirold et al., 2001 Mirold et al., 2001 Mirold et al., 2001 Mirold et al., 2001 Mirold et al., 2001 Kaniga et al., 1994
1 2
Bacterial invasion into tissue culture cells was determined in Gentamicin protection assays. The cytotoxicity was determined by infection of macrophages with the mutant strains, staining with ethidium homodimer and examing for nuclear staining by fluorescence microscopy. 3 independent experiments indicate that the experimental error is in the range of 10% of the given value. (reproduced with permission from (Mirold et al., 2001)).
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count for the residual invasiveness observed with the sopE, sopE2, sopB mutant. In conclusion, host cell invasion by Typhimurium strains is mediated by the SPI1 type III secretion apparatus and effector proteins encoded within this locus (SipA, SipC) and elsewhere in the chromosome (SopB, SopE, SopE2; Fig. 1). Together, these loci are referred to as an “invasion virulon” to emphasize the direct functional requirement of different loci from distant regions of the chromosome (Mirold et al., 2001). The regulatory circuits ensuring coordinate expression of these loci are just beginning to be understood (Darwin and Miller, 2001).
Evolution of Salmonella host cell invasion Usually, virulence genes found in pathogens but absent from non-pathogenic relatives, are clustered in “pathogenicity islands” (PAI). PAI are genetic elements that were acquired by horizontal gene transfer and which encode all functions necessary to provide the bacterium with a novel virulence characteristic (reviewed by Hacker et al. (1997)). The chromosomal distribution of the Salmonella genes mediating host cell invasion is quite different (Fig. 1). Therefore, it was of interest to study the evolution of Salmonella host cell invasion in more detail. Phylogenetic analyses had shown that the genes for the SPI1 type III secretion apparatus had been acquired in the very early phase of Salmonella evolution, soon after the divergence from the Escherichia lineages (Boyd et al., 1997; Li et al., 1995). Southern hybridization analysis with gene probes for the effector proteins encoded within SPI1 demonstrated that sipA, sipB, sipC and sptP are present in all strains of S. bongori and the different S. enterica subspecies tested (Tab. 3) (Mirold et al., 2001). This argues that the genes for these effector proteins had probably been acquired early on together with the genes of the SPI1 type III secretion apparatus. Surprisingly, sopB and sopE2 (as well as sopD) were also present in all Salmonella strains tested (Tab. 3). Table 3. Distribution of genes for SPI1-dependent effector proteins.
Phylogenetic analyses of the coding sequences confirmed that these genes had been acquired during the first phase of Salmonella evolution before divergence of S. bongori and the S. enterica subspecies (Fig. 2). Since translocation of SopB and SopE2 is sufficient to allow fairly efficient host cell invasion (Tab. 2), the last common ancestor of all contemporary Salmonella lineages was probably invasive, already. Figure 3 summarizes our current knowledge of the ability of the last common ancestor of all contemporary Salmonella lineages to interfere with host cellular signalling and to mediate invasion. SopE2 acts as a Gnucleotide exchange factor (GEF) for host cellular Cdc42 (Stender et al., 2000). Active, GTP-bound Cdc42 triggers signalling events leading to expression of pro-inflammatory cytokines and the formation of membrane ruffles. The latter process, which probably involves WASP, leads to recruitment of the Arp2/3 complex to the sites of bacterial contact (Stender et al., 2000). The Arp2/3 complex is the only eukaryotic factor known to catalyze de novo actin polymerization. The exact role of SopB is less clear. It has been shown that SopB is a PI phosphatase (Norris et al., 1998) and that this catalytic activity contributes to the reorganization of the actin cytoskeleton (Zhou et al., 2001). Further work will be required to clarify how altered PI phosphate levels affect the bacterial entry mechanism. As discussed above, other effectors that had been present in the last common ancestor of all contemporary Salmonella lineages (namely SipA, possibly SipC) may also have contributed to host cell invasion by binding and stabilization (or de novo formation) of actin structures (Fig. 3). Our phylogenetic analyses have shown that this ancient signalling capacity is well conserved among the contemporary Salmonella lineages. However, in certain strains we have identified alterations. For example, Typhi strains which harbor a defective sopE2 gene seem to employ sopE instead (Stender et al., 2000; Prager et al., 2000). SopE is about 70 % identical to SopE2 and is only present in certain Salmonella strains. It has been introduced into the Salmonella population only recently (Mirold et al., 2001). In sopE-positive Typhimurium strains it is encoded in the tail/tail-fiber region of the temperate P2-like prophage SopEΦ (Hardt et al., 1998b; Mirold et al., 1999). Interestingly, most of the SopEΦ lysogenic Typhimurium isolates belong to one strain that caused a major epidemic in England and the former East Germany during the 70’s (Mirold et al., 1999). Therefore, acquisition of a sopE allele by lysogenic conversion with SopEΦ might have been one of the factors leading to the emergence of this epidemic strain. In fact, SopEΦ lysogens of the Typhimurium strain ATCC14 028 were about 2.5-fold more invasive than the isogenic wild-type strain (Mirold and Hardt,
S. typhimurium SPI1 type III effector proteins
483
Fig. 2. Schematic representation of the evolution of host cell invasion by Salmonella spp. (reproduced with permission from (Mirold et al., 2001)).
unpublished). In addition, several effector proteins translocated via the S. enterica SPI2 type III secretion system are also variably distributed between different strains and are associated with bacteriophages (Figueroa-Bossi et al., 2001; Miao et al., 1999; Miao and Miller, 2000). Overall, these observations support the hypothesis that variable assortment of the effector protein repertoire by horizontal gene transfer might be a driving force for the emergence of new epidemic strains.
Functional differences between SopE and SopE2 At first glance SopE and SopE2 look very much alike: Both are 240 amino acids in length; they share about 70 % identical amino acid residues; both are translocated into host cells via the SPI1 type III secretion system and both act as efficient G-nucleotide exchange factors (GEF) for host cellular Cdc42 (Stender et al., 2000). However, the data discussed above indicate that acquisition of sopE by lysogenic conversion with SopEΦ might alter the virulence of a sopE2+ Typhimurium strain. This effect might simply be due to an increased “sopE/E2 gene dosage”. Alternatively, there might be functional differences between SopE and SopE2. Indeed, in vitro binding and G-nucleotide exchange assays using purified SopE and SopE2 proteins revealed that SopE interacts efficiently with Cdc42 and Rac1, while SopE2 only interacts efficiently with Cdc42 but not with Rac1 (Friebel and Hardt, unpublished). These differences in substrate specificity can also be observed in tissue culture cell infection experiments: Infection of tissue culture cells with a Typhimurium strain which only translocates SopE (but not SopE2 or SopB) results in specific activation of host cellular Cdc42 and Rac1, and infection of HUVEC cells leads to pronounced lamellipodia formation. In contrast, a Typhimurium strain translocating SopE2 (but not SopE or SopB) only activates Cdc42, but not
Fig. 3. Summary of the assumed abilities of the last common ancestor of all contemporary Salmonella lineages to interfere with host cellular signalling leading to transcriptional activation and membrane rearrangements mediating invasion of Salmonella spp. into the host cell.
Rac1. Infection of HUVEC cells with the latter strain leads to formation of filopodia but only few lamellipodia (Friebel and Hardt, unpublished). This indicates that SopE and SopE2 have different substrate specificities in vivo. Typhimurium strains just harboring a sopE2 allele are only capable of directly activating Cdc42, but not Rac1. However, acquisition of a sopE gene by lysogenic conversion with SopEΦ can alter the signalling capacity of this strain, allowing direct activation of Cdc42 and Rac1. It is tempting to speculate that the altered capacity to modulate host cellular signalling pathways might be one of the factors explaining the increased epidemic virulence of SopEΦ lysogens. Future work will have to prove this theory by analyzing the virulence of the lysogens in appropriate animal models. Acknowledgements. We are grateful to W. Rabsch, H. Tschäpe and M. Aepfelbacher for fruitful discussions. Work in the lab of W.-D. Hardt is supported by grants from the Deutsche Forschungsgemeinschaft and the Volkswagen-Stiftung.
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Int. J. Med. Microbiol. 291, 479-485 (2002) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ijmm
Characterization of effector proteins translocated via the SPI1 type III secretion system of Salmonella typhimurium Kristin Ehrbar, Susanne Mirold, Andrea Friebel, Silke Stender, Wolf-Dietrich Hardt Max von Pettenkofer-Institut, Pettenkoferstraße 9a, D-80336 München, Germany
Abstract Salmonella spp. employ a conserved type III secretion system encoded within the pathogenicity island 1 (SPI1; centisome 63) to translocate effector proteins into the host cytosol. The translocated effector proteins trigger diverse responses including bacterial internalization. In a mutation analysis we have defined the set of effector proteins mediating tissue culture cell invasion. This set includes sopE2 (centisome 40–42), sopB (SPI5, centisome 20) and in the case of S. typhimurium SL1344 also the phage-encoded effector sopE (SopEΦ, centisome 59–60). A triple mutant SL1344 derivative deficient of SopE, SopE2 and SopB was more than 100-fold attenuated in tissue culture cell invasion. Phylogenetic analyses indicate that the last common ancestor of all contemporary Salmonella lineages already harbored all genes necessary for host cell invasion, namely the SPI1 type III secretion system, sopE2 and sopB. SopE, which is 70 % identical to sopE2 is only present in some Salmonella strains and emerged later well after the divergence of the contemporary Salmonella lineages. Interestingly, S. typhimurium strains that harbor sopE are associated with epidemics, arguing that sopE is one of the factors determining the “fitness” of a strain. We found that SopE can specifically activate a different set of host cellular RhoGTPases than SopE2. This allows the bacteria to fine tune host cellular responses very precisely and may offer an explanation for the improved epidemic fitness of sopE-positive S. typhimurium strains. Key words: Salmonella – type III secretion – evolution – phage – Cdc42
Introduction Infections with Salmonella spp. are among the leading causes of bacterial enteritis in developed countries. The type of disease can range from a mild gastroenteritis to systemic infection and is determined by the virulence characteristics of the Salmonella strain as well as by the host species. Detailed phylogenetic analysis has revealed that Salmonella spp. have diverged from the Escherichia spp. about 100–160 million years ago (Ochman and Wilson, 1987). S. enterica harbors about 400–800 kb of DNA including at least 5 separate ‘pathogenicity islands’ necessary for disease progression which are absent from the E. coli genome (reviewed by
Groisman and Ochman (1996)). It is thought that much of the additional DNA acquired via horizontal gene transfer has played a role in the evolution of Salmonella spp. as pathogens (Ochman and Moran, 2001). Two of these additional DNA segments termed Salmonella pathogenicity islands 1 and 2 (SPI1 and SPI2) encode type III secretion systems (Hueck, 1998). Type III secretion systems have been identified in many gram-negative bacteria living in close association with animals or plants (Galán and Collmer, 1999). Sequence similarities and evidence from electron microscopy studies suggest that type III secretion systems have evolved from bacterial flagella systems. They allow to inject (translocate) bacterial (effector) proteins directly
Corresponding author: Wolf-Dietrich Hardt, Max von Pettenkofer-Institut, Pettenkoferstraße 9a, D-80336 München, Germany, Phone: +49 89 5160 5263, Fax: +49 89 5160 5223, E-mail: [email protected] 1438-4221/01/291/6-7-479 $ 15.00/0
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into the cytosol of host cells to manipulate host cellular signalling cascades. Acquisition of SPI1 has been a key event in the early phase of the evolution of Salmonella as a pathogen (Groisman and Ochman, 1996) and the genes for the SPI1 type III secretion apparatus are present in all contemporary Salmonella lineages (Boyd et al., 1997; Li et al., 1995). Work on strains of S. enterica subspecies 1 serotypes Typhimurium and Dublin has shown that the SPI1 type III secretion system mediates penetration of the host ileal mucosa and the induction of PMN transmigration and diarrhea in the bovine ileum (reviewed by Wallis and Galyov (2000)). In tissue culture cells proteins translocated by the SPI1 type III secretion system induce cell death in macrophages, chloride secretion, IL8 production, membrane ruffling and bacterial entry into non-phagocytic cell lines (reviewed by Galán and Zhou (2000)). So far, 12 different translocated effector proteins are known to be translocated into host cells via the SPI1 type III secretion system (Tab. 1). Studying the function of each of these effector proteins in Salmonella virulence has proven to be quite difficult, since disruption of genes for a single translocated effector protein has often resulted only in minor virulence deTable 1. Effector proteins translocated via the SPI1 type III secretion system. Effector
Location
(Proposed) molecular function
Reference
SipA
SPI1
F-actin stabilization and bundling enhances SipC-mediated actin polymerization?
Zhou et al., 1999a,b McGhie et al., 2001
SipB#
SPI1
cell death in macrophages
Hersh et al., 1999
SipC#
SPI1
actin polymerization/ bundling
McGhie et al., 2001 Hayward and Koronakis, 1999
SptP
SPI1
GAP for RhoGTPases
Fu and Galan, 1999
AvrA
SPI1
?
Hardt et al., 1997
SopA
cs 41-42
?
Wood et al., 2000
SopB
SPI5, cs 20
PI phosphatase
Galyov et al. 1997
SopE
SopEF cs 60
GEF for RhoGTPases
Hardt et al., 1998b Wood et al., 1996
SopE2
cs 40-42
GEF for RhoGTPases
Stender et al., 2000
SopD
cs 64
?
Jones et al., 1998
SspH1*
GIFSY-3 cs 23-25
?
Miao et al., 1999 Figueroa-Bossi et al., 2001
SlrP*
cs 18-19
?
Tsolis et al., 1999
* also translocated via the SPI2 type III secretion system. # also involved in translocation of the other effector proteins (Collazo and Galan, 1997).
fects (see Table 1). Recent work using Typhimurium mutants lacking multiple effector proteins suggests that this might be due to functional redundancy between different translocated effector proteins (Jones et al., 1998; Stender et al., 2000; Zhou et al., 2001; Mirold et al., 2001). The identification and characterization of the translocated effector proteins have also been complicated by the fact that many key effector proteins are not encoded within SPI1 but in distant regions of the chromosome (Fig. 1). In this article we will review the properties of Salmonella effector proteins translocated via the SPI1 type III secretion system with a focus on those effector proteins mediating host cell invasion.
Identification of the effector proteins mediating host cell invasion Soon after the discovery of the first genes encoding functional components of the SPI1 type III secretion apparatus it was found that it is involved not only in the early gut-associated stages of the murine Typhimurium infection but also in the triggering of host cytoskeletal rearrangements and tissue culture cell invasion (reviewed by Galán (1999)). The translocated effector proteins triggering these responses have been identified by subsequent genetic analyses and by sequencing of proteins that were secreted by wild-type Salmonella strains but not by mutants with a disrupted type III secretion apparatus (Hueck et al., 1995; Kaniga et al., 1995a and b; Wood et al., 1996). However, considerable efforts were still required to unequivocally identi-
Fig. 1. Chromosomal map of S. enterica subspecies I serovar Typhimurium. The locations of SPI1 and of the known effector protein genes within and outside SPI1 are shown.
S. typhimurium SPI1 type III effector proteins
fy those effector proteins which modulate host cellular signalling necessary for invasion. Typhimurium strains carrying single mutations in genes encoding secreted proteins could be grouped into three categories: 1. Secreted proteins which are part of the secretion apparatus and absolutely required for the secretion of all other secreted proteins. SpaO and invJ mutants are non-invasive (Collazo and Galán, 1996). 2. Secreted proteins required for delivery of proteins of the 3rd group into the host cell. SipB, sipC and sipD mutants still secrete the other proteins into the culture media, but they are non-invasive (Collazo and Galán, 1997). However, it should be pointed out that SipB and SipC might serve dual functions acting as “translocases” but also as effector proteins once they arrive in the host cytosol. 3. Genuine effector proteins that are not required for secretion or translocation of other proteins. This includes SipA, SptP, AvrA, SopB, SopD, SopE, SopE2 and probably also SopA, SlrP and SspH1. Mutation analyses revealed that only sopB, sopE and sopE2 mutants were mildly (about 2-fold) attenuated in tissue culture cell invasion (Wood et al., 1996; Hardt et al., 1998a; Hong and Miller, 1998; Wood et al., 1998; Bakshi et al., 2000; Stender et al., 2000). These defects were much weaker than the defects (500-fold attenuation) observed with Salmonella mutants harboring an inactivated SPI1 type III translocation apparatus. Biochemical analyses demonstrated that SopE and SopE2 act as G-nucleotide exchange factors for host cellular RhoGTPases like Cdc42 (Hardt et al., 1998a; Stender et al., 2000). RhoGTPases are key regulators of diverse cellular activities including the expression of pro-inflammatory cytokines and the structural organization of the eukaryotic actin cytoskeleton. Activation of Cdc42 and Rac1 is known to lead to membrane ruffling (reviewed by Bishop and Hall (2000)). SopB
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harbors two consensus motifs for eukaryotic inositol polyphosphate 4-phosphatases and alters PI phosphate levels inside infected host cells (Norris et al., 1998; Zhou et al., 2001; Feng et al., 2001). Transfection experiments with SopE, SopE2 or SopB expression vectors demonstrated that each of these effector proteins is sufficient to induce dramatic cytoskeletal rearrangements in tissue culture cells or mediate bacterial internalization (Hardt et al., 1998a; Stender et al., 2000; Zhou et al., 2001). This evidence suggested that SopE, SopE2 and SopB have at least partially redundant functions and that they cooperate to mediate efficient host cell invasion. Indeed, double mutants of Typhimurium SL1344 lacking two of the three effector proteins are 4–10-fold less invasive than the isogenic wild-type strain, and a triple mutant with disrupted genes for SopE, SopE2 and SopB is virtually unable to enter host cells (Tab. 2). This defect could be complemented with SopE or SopE2 expression vectors and to a significant extent also with a SopB expression vector. Together, these observations argue that SopE, SopE2 and SopB jointly mediate host cell invasion. Other translocated effector proteins might also contribute to invasion. Indeed, a sipA mutant is somewhat less invasive and SipA can directly bind to actin, stabilizes F-actin and modulates the bundling of F-actin filaments (Zhou et al., 1999a, b). However, induction of cytoskeletal rearrangements by SipA alone has not been demonstrated. In vitro experiments show that purified (refolded) SipC can mediate de novo actin polymerisation, and microinjection of purified SipC leads to dramatic rearrangements in the actin distribution in tissue culture cells. In addition it has been found that SipA cooperates with SipC in remodelling actin structures in semi-permeabilized cells (McGhie et al., 2001). Taken together, these observations argue that some effector function of SipC and/or SipA might ac-
Table 2. Several effector proteins cooperate to mediate host cell invasion. Strain
Relevant phenotype
Invasiveness (%)1
Macrophage cytotoxicity2 (%)
Reference
SL1344 SB856 M202 M516 M511 M516 + pM149 M516 + pSB1130 M516 + pM515 SB161
wild-type sopE sopE, sopE2 sopE, sopE2, sopB sopE, sopE2, sopB, sopD sopE, sopE2, sopB, (psopE2) sopE, sopE2, sopB, (psopE) sopE, sopE2, sopB, (psopB) invG
100 53.3 29 28.4 3.9 .95 1.0 .93 0.4 80.8 31 213.0 81 12.2 1.2 .17 0.1
100 94 86 90 85 87 90 17 3
Hoiseth and Stocker, 1981 Hardt et al., 1998a Stender et al., 2000 Mirold et al., 2001 Mirold et al., 2001 Mirold et al., 2001 Mirold et al., 2001 Mirold et al., 2001 Kaniga et al., 1994
1 2
Bacterial invasion into tissue culture cells was determined in Gentamicin protection assays. The cytotoxicity was determined by infection of macrophages with the mutant strains, staining with ethidium homodimer and examing for nuclear staining by fluorescence microscopy. 3 independent experiments indicate that the experimental error is in the range of 10% of the given value. (reproduced with permission from (Mirold et al., 2001)).
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count for the residual invasiveness observed with the sopE, sopE2, sopB mutant. In conclusion, host cell invasion by Typhimurium strains is mediated by the SPI1 type III secretion apparatus and effector proteins encoded within this locus (SipA, SipC) and elsewhere in the chromosome (SopB, SopE, SopE2; Fig. 1). Together, these loci are referred to as an “invasion virulon” to emphasize the direct functional requirement of different loci from distant regions of the chromosome (Mirold et al., 2001). The regulatory circuits ensuring coordinate expression of these loci are just beginning to be understood (Darwin and Miller, 2001).
Evolution of Salmonella host cell invasion Usually, virulence genes found in pathogens but absent from non-pathogenic relatives, are clustered in “pathogenicity islands” (PAI). PAI are genetic elements that were acquired by horizontal gene transfer and which encode all functions necessary to provide the bacterium with a novel virulence characteristic (reviewed by Hacker et al. (1997)). The chromosomal distribution of the Salmonella genes mediating host cell invasion is quite different (Fig. 1). Therefore, it was of interest to study the evolution of Salmonella host cell invasion in more detail. Phylogenetic analyses had shown that the genes for the SPI1 type III secretion apparatus had been acquired in the very early phase of Salmonella evolution, soon after the divergence from the Escherichia lineages (Boyd et al., 1997; Li et al., 1995). Southern hybridization analysis with gene probes for the effector proteins encoded within SPI1 demonstrated that sipA, sipB, sipC and sptP are present in all strains of S. bongori and the different S. enterica subspecies tested (Tab. 3) (Mirold et al., 2001). This argues that the genes for these effector proteins had probably been acquired early on together with the genes of the SPI1 type III secretion apparatus. Surprisingly, sopB and sopE2 (as well as sopD) were also present in all Salmonella strains tested (Tab. 3). Table 3. Distribution of genes for SPI1-dependent effector proteins.
Phylogenetic analyses of the coding sequences confirmed that these genes had been acquired during the first phase of Salmonella evolution before divergence of S. bongori and the S. enterica subspecies (Fig. 2). Since translocation of SopB and SopE2 is sufficient to allow fairly efficient host cell invasion (Tab. 2), the last common ancestor of all contemporary Salmonella lineages was probably invasive, already. Figure 3 summarizes our current knowledge of the ability of the last common ancestor of all contemporary Salmonella lineages to interfere with host cellular signalling and to mediate invasion. SopE2 acts as a Gnucleotide exchange factor (GEF) for host cellular Cdc42 (Stender et al., 2000). Active, GTP-bound Cdc42 triggers signalling events leading to expression of pro-inflammatory cytokines and the formation of membrane ruffles. The latter process, which probably involves WASP, leads to recruitment of the Arp2/3 complex to the sites of bacterial contact (Stender et al., 2000). The Arp2/3 complex is the only eukaryotic factor known to catalyze de novo actin polymerization. The exact role of SopB is less clear. It has been shown that SopB is a PI phosphatase (Norris et al., 1998) and that this catalytic activity contributes to the reorganization of the actin cytoskeleton (Zhou et al., 2001). Further work will be required to clarify how altered PI phosphate levels affect the bacterial entry mechanism. As discussed above, other effectors that had been present in the last common ancestor of all contemporary Salmonella lineages (namely SipA, possibly SipC) may also have contributed to host cell invasion by binding and stabilization (or de novo formation) of actin structures (Fig. 3). Our phylogenetic analyses have shown that this ancient signalling capacity is well conserved among the contemporary Salmonella lineages. However, in certain strains we have identified alterations. For example, Typhi strains which harbor a defective sopE2 gene seem to employ sopE instead (Stender et al., 2000; Prager et al., 2000). SopE is about 70 % identical to SopE2 and is only present in certain Salmonella strains. It has been introduced into the Salmonella population only recently (Mirold et al., 2001). In sopE-positive Typhimurium strains it is encoded in the tail/tail-fiber region of the temperate P2-like prophage SopEΦ (Hardt et al., 1998b; Mirold et al., 1999). Interestingly, most of the SopEΦ lysogenic Typhimurium isolates belong to one strain that caused a major epidemic in England and the former East Germany during the 70’s (Mirold et al., 1999). Therefore, acquisition of a sopE allele by lysogenic conversion with SopEΦ might have been one of the factors leading to the emergence of this epidemic strain. In fact, SopEΦ lysogens of the Typhimurium strain ATCC14 028 were about 2.5-fold more invasive than the isogenic wild-type strain (Mirold and Hardt,
S. typhimurium SPI1 type III effector proteins
483
Fig. 2. Schematic representation of the evolution of host cell invasion by Salmonella spp. (reproduced with permission from (Mirold et al., 2001)).
unpublished). In addition, several effector proteins translocated via the S. enterica SPI2 type III secretion system are also variably distributed between different strains and are associated with bacteriophages (Figueroa-Bossi et al., 2001; Miao et al., 1999; Miao and Miller, 2000). Overall, these observations support the hypothesis that variable assortment of the effector protein repertoire by horizontal gene transfer might be a driving force for the emergence of new epidemic strains.
Functional differences between SopE and SopE2 At first glance SopE and SopE2 look very much alike: Both are 240 amino acids in length; they share about 70 % identical amino acid residues; both are translocated into host cells via the SPI1 type III secretion system and both act as efficient G-nucleotide exchange factors (GEF) for host cellular Cdc42 (Stender et al., 2000). However, the data discussed above indicate that acquisition of sopE by lysogenic conversion with SopEΦ might alter the virulence of a sopE2+ Typhimurium strain. This effect might simply be due to an increased “sopE/E2 gene dosage”. Alternatively, there might be functional differences between SopE and SopE2. Indeed, in vitro binding and G-nucleotide exchange assays using purified SopE and SopE2 proteins revealed that SopE interacts efficiently with Cdc42 and Rac1, while SopE2 only interacts efficiently with Cdc42 but not with Rac1 (Friebel and Hardt, unpublished). These differences in substrate specificity can also be observed in tissue culture cell infection experiments: Infection of tissue culture cells with a Typhimurium strain which only translocates SopE (but not SopE2 or SopB) results in specific activation of host cellular Cdc42 and Rac1, and infection of HUVEC cells leads to pronounced lamellipodia formation. In contrast, a Typhimurium strain translocating SopE2 (but not SopE or SopB) only activates Cdc42, but not
Fig. 3. Summary of the assumed abilities of the last common ancestor of all contemporary Salmonella lineages to interfere with host cellular signalling leading to transcriptional activation and membrane rearrangements mediating invasion of Salmonella spp. into the host cell.
Rac1. Infection of HUVEC cells with the latter strain leads to formation of filopodia but only few lamellipodia (Friebel and Hardt, unpublished). This indicates that SopE and SopE2 have different substrate specificities in vivo. Typhimurium strains just harboring a sopE2 allele are only capable of directly activating Cdc42, but not Rac1. However, acquisition of a sopE gene by lysogenic conversion with SopEΦ can alter the signalling capacity of this strain, allowing direct activation of Cdc42 and Rac1. It is tempting to speculate that the altered capacity to modulate host cellular signalling pathways might be one of the factors explaining the increased epidemic virulence of SopEΦ lysogens. Future work will have to prove this theory by analyzing the virulence of the lysogens in appropriate animal models. Acknowledgements. We are grateful to W. Rabsch, H. Tschäpe and M. Aepfelbacher for fruitful discussions. Work in the lab of W.-D. Hardt is supported by grants from the Deutsche Forschungsgemeinschaft and the Volkswagen-Stiftung.
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