Salmonella pathogenicity islands: big virulence in small packages

Microbes and Infection, 2, 2000, 145−156 © 2000 Éditions scientifiques et médicales Elsevier SAS. All rights reserved

Review

Salmonella pathogenicity islands: big virulence in small packages Sandra L. Marcus, John H. Brumell, Cheryl G. Pfeifer, B. Brett Finlay* Biotechnology Laboratory, and Departments of Biochemistry & Molecular Biology and Microbiology & Immunology, University of British Columbia, Wesbrook Building 237, 6174 University Boulevard Vancouver, BC, V6T 1Z3, Canada

ABSTRACT – Reflecting a complex set of interactions with its host, Salmonella spp. require multiple genes for full virulence. Many of these genes are found in ‘pathogenicity islands’ in the chromosome. Salmonella typhimurium possesses at least five such pathogenicity islands (SPI), which confer specific virulence traits and may have been acquired by horizontal transfer from other organisms. We highlight recent progress in characterizing these SPIs and the function of some of their genes. The role of virulence genes found on a highly conserved plasmid is also discussed. Collectively, these packages of virulence cassettes are essential for Salmonella pathogenesis. © 2000 Éditions scientifiques et médicales Elsevier SAS Salmonella spp. / pathogenicity island / virulence / diarrhoea / typhoid fever / type III secretion

1. Introduction Salmonella is a facultative intracellular pathogen which, depending on the serotype and host, can cause diseases ranging from gastroenteritis to typhoid fever. For example, Salmonella enterica serovar typhimurium (S. typhimurium), which initiates disease normally limited to gastroenteritis in humans, causes systemic disease in mice and has been used as an animal model of typhoid fever. Salmonella infections are usually acquired by ingestion of contaminated food or water. In systemic (typhoid-like) disease, following ingestion, the bacteria survive the acid pH of the stomach, colonize the Peyer’s patches of the intestine, and penetrate the gut barrier via M cells (specialized epithelial cells). From there, they disseminate to the local mesenteric lymph nodes and then to the spleen and liver via phagocytic cells [1, 2]. S. typhimurium invade cultured epithelial and macrophage cells in vitro and remain inside a unique membrane-bound vacuole, the Salmonella-containing vacuole (SCV), where they begin to replicate. Mutants that are defective for invasion or replication are also attenuated in mice. For most bacterial pathogens, virulence requires multiple factors [3]. It has been estimated that approximately 4% of the S. typhimurium genome is required for fatal infection of mice, which translates into over 200 virulence genes [4]. Moreover, 2-dimensional electrophoresis of S. typhimurium proteins recovered from infected intestinal

* Correspondence and reprints Microbes and Infection 2000, 145-156

epithelial, macrophage, and murine liver cell lines revealed the presence of many proteins specific to infection of each cell type [5, 6]. The need for so many virulence determinants is thought to reflect the complex interactions of salmonella with the infected host [1]. Furthermore, bacterial survival in the host appears to result from a delicate balance of many gene products acting at the correct time in the correct location [4]. These genes are found on plasmids or within the chromosome as units of one, or a few of, virulence genes (islets) or large cassettes composed of a series of genes and operons (pathogenicity islands). Pathogenicity islands typically accommodate large clusters of genes that contribute to a particular virulence phenotype, which is generally manifested at a specific time during the course of infection. Thus, for many enteric bacteria, a single pathogenicity island can convert a normally benign microorganism into a pathogen. For example, a 35-kb region found on the enteropathogenic Escherichia coli (EPEC) chromosome, termed the locus of enterocyte effacement (LEE), is responsible for the formation of attaching and effacing lesions on intestinal epithelial cells. The LEE region contains a type III secretion system, which includes all the machinery necessary to deliver virulence proteins to the host cell (see below). The ability to induce the formation of attaching and effacing lesions can be conferred to a laboratory strain of E. coli by a plasmid carrying the LEE region [7]. These observations suggest that nonpathogenic E. coli, which are normal residents of the human intestine, have the potential to become pathogenic if they acquire a particular virulence gene cluster, illustrating the possible evolutionary 145

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emergence of novel pathogens. Conversely, the acquisition of a pathogenicity island by an organism does not guarantee its transformation into a pathogen [8]. Rather, virulence is also dependent on the recipient microorganism and host. For example, nonpathogenic Listeria seeligeri harbours most known virulence genes within a 10-kb region that is also present in pathogenic species of Listeria (L. monocytogenes and L. ivanovii) [9]. This review highlights recent progress in our understanding of genes required for Salmonella virulence, but which are absent from closely related benign microorganisms such as E. coli (K-12). Although some of these genes are found on a virulence plasmid common to many Salmonella serovars, most are encoded within pathogenicity islands [8]. Salmonella pathogenicity islands (SPIs) are defined as large gene cassettes within the Salmonella chromosome that encode determinants responsible for establishing specific interactions with the host, and are required for bacterial virulence in a given animal model. Like other pathogenicity islands, SPIs generally have a GC content lower (between 37 and 47%) than the rest of the bacterial chromosome (about 52%) and are often inserted into tRNA genes. Therefore, SPIs have likely been acquired by horizontal transfer from phage or plasmids of unknown origin, and they are highly conserved between the different Salmonella serotypes. A total of five SPIs have been identified thus far (figure 1A). Salmonella pathogenicity island 1 (SPI1) is primarily required for bacterial penetration of the epithelial cells of the intestine (invasion), while SPI2, 3, and 4 are primarily required for growth and survival of bacteria within the host, manifested in the systemic phase of disease. Recently identified virulence factors encoded by SPI5 appear to mediate the inflammation and chloride secretion which characterize the enteric phase of disease. Finally, genes on the virulence plasmid may be required for growth of the bacteria within host macrophages and are needed for prolonged survival in mice. Interestingly, SPI1 and SPI2 both encode type III secretion systems, which mediate the respective virulence phenotype by translocating bacterially-encoded proteins into the host cell cytosol.

2. Type III secretion systems Type III secretion systems are used by many bacterial pathogens to deliver virulence factors to the host cell and interfere with or subvert normal host cell signalling pathways. Several excellent reviews have been written on this topic [10, 11]. For example, Yersinia pseudotuberculosis inhibits its own uptake into macrophages by injecting YopH, a phosphatase that interferes with the signalling required for receptor-mediated endocytosis and phagocytosis [12]. In contrast, the secreted proteins encoded by the Shigella spp. virulence plasmid and SPI1 induce cytoskeletal rearrangements in the host cell that lead to bacterial invasion into nonphagocytic epithelial cells (see below). Type III secretion systems consist of many components, including more than twenty proteins, some of which are homologous to those involved in flagellar 146

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assembly. Effector proteins generally require specific chaperones which prevent incorrect folding, degradation and premature association, and may even aid delivery of the effector into host cells. These systems are highly regulated, and proteins are only secreted when the bacteria sense specific environmental cues. Type III secretion systems have been reported to be contact-dependent for Yersinia and Shigella spp. Of the five SPIs described so far, SPI1 and SPI2 each encode distinct type III secretion systems. Whether SPI1 type III secretion is also contactdependent is still controversial. In vitro, the conditions vary in which Yersinia, Shigella, and SPI1 [13] type III secretion systems can be induced to secrete in the absence of host cells.

3. SPI1 SPI1 is the best-characterized of the five SPIs. It is approximately 40 kb in size and is located at 63 centisomes (cs) on the S. typhimurium chromosome. SPI1 is flanked by fhlA and mutS and has an overall GC content of 42%. SPI1 contains at least 29 genes (figure 1B), encoding various components of a type III secretion system, its regulators and its secreted effectors [14]. The virulence phenotype associated with SPI1 is dependent on the ability of its encoded secretion system to deliver effector proteins (encoded both inside and outside of SPI1, see below) into the host cell cytosol. By delivery of these effectors, SPI1 enables S. typhimurium to efficiently penetrate the intestinal epithelium. This is evidenced by the finding that SPI1 mutants (e.g., invA, orgA, see table I) are attenuated for virulence when inoculated orally, but not systemically, in mice [15, 16]. This suggests that the role of SPI1, which was acquired early in the evolution of S. enterica spp. [3], may be limited to colonization of the gut and that other virulence determinants were later acquired to allow for establishment of systemic disease. Electron microscopy studies of the SPI1 secretion system have revealed supramolecular structures which span the inner and outer membranes, resembling needles that project from the surface of the bacterium [17]. These structures were biochemically purified and found to contain several components of the SPI1 type III secretion system, including InvG, PrgH and PrgK. Expression of an epitope tagged form of PrgH allowed immunoelectron microscopy identification of the needle complexes, confirming that these structures were indeed the type III secretion system encoded by SPI1. It seems likely that these needle-like structures are directly involved in the translocation process by ‘injecting’ virulence effectors into the host cell cytosol. The translocation of effectors via the SPI1 encoded type III secretion system allows Salmonella spp. to enter nonphagocytic cells, such as M cells, of the intestinal epithelium [11, 18]. The invasive process involves massive ruffling of the host cell plasma membrane at the site of interaction with the bacterium and bacterial uptake into large vesicles resembling macropinosomes [19, 20]. Crucial for invasion are rearrangements of the host cell actin cytoskeleton, witnessed by the ability of cytochalasins to Microbes and Infection 2000, 145-156

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B

Figure 1. A. Schematic representation of the S. typhimurium chromosome, which is divided into 100 cs. Known SPIs are shown on the outside of the chromosomal circle, while a number of other virulence genes and transcriptional regulators discussed in the text are shown on the inside. B. Schematic representation of SPIs. inhibit S. typhimurium uptake [21]. It should be noted that invasion of S. typhimurium is both morphologically and biochemically unique from receptor-mediated phagocytosis by professional phagocytes (reviewed in [22]). Importantly, invasion is mediated by the delivery of effectors that directly engage host cell signalling pathways. Microbes and Infection 2000, 145-156

The regulation of SPI1 gene expression is complex, and is beyond the scope of this review. We refer the reader to two recent papers which summarize the current knowledge in this area [23, 24]. In vitro, many factors regulate the ability of S. typhimurium to invade cells, including oxygen, osmolarity and growth phase of the bacteria [14]. 147

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Table I. Selected S. typhimurium virulence factors associated with pathogenicity islands. Location

Gene

Type

Biochemical activity

Effect on host cell

Mutant phenotype

Reference

SPI (b3c5)

invA

Structural (inner membrane)

Translocation

?

Translocation?

sptP

Translocated effector

Tyrosine phosphatase

sipA

Translocated effector

Binds actin, activates plastin

Actin rearrangements and bundling

LD50 attenuated after oral infection but not intraperitoneal. Non-invasive in vitro Same as invA. Regulation of orgA by low oxygen levels Invasion unaffected but attenuated in colonization of spleen in mice Invasion slightly attenuated in vitro, loss of actin polymerization in host cell at site of entry

[15]

orgA

Delivery of type III secreted effectors Delivery of type III secreted effectors Actin rearrangements?

sipB

Translocated effector

Apoptosis of macrophages

[31]

sipC

Translocated effector?

Translocation, caspase 1 activation Translocation, other?

[33]

sopE*

Translocated effector

Delivery of type III secreted effectors, other? Actin rearrangements, cytokine production Delivery of type III secreted effectors?

61 cs

SPI2 (31 cs) ssaJ

YscJ/MxiJ/PrgK family of lipoproteins (structural?)

Activates Cdc42 and Rac GTPases Translocation?

sseABC

Translocated effectors?

?

?

spiC

Translocated effector

Inhibits endosome-endosome fusion in vitro

SPI3 (82 cs) mgtCB

Cation transporters?

Mg2+ uptake, others?

Inhibits fusion of SCV with lysosomes and endosomes. Interferes with normal trafficking of the transferrin receptor ?

SPI4 (92 cs) Various SPI5 (25 cs) sopB/sigD*

Type I Secretion? Others? Translocated effector

Toxin delivery? Apoptosis? Inositol phosphate phosphatase Chloride secretion

pipB

[16] [25, 26] [29, 30]

Invasion slightly attenuated in vitro. Virulence unaffected in vivo Virulence attenuated in mice and bacteria unable to spread to mesenteric lymph nodes. Mutants unable to replicate in the spleen. Virulence attenuated in mice. Mutants unable to replicate in macrophages in vitro. Attenuated virulence in mice and survival in macrophages in vitro

[37, 39]

Attenuated virulence in mice and survival in macrophages in vitro Intramacrophage survival Attenuated enteropathogenesis in ileal loop model Attenuated virulence in mice

[57, 58]

[44, 47] [46] [51]

[61, 66] [70, 71] [53]

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* Note: Translocated via the SPI1 type III secretion system into the host cell cytosol.

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The main factor regulating SPI1 secretion appears to be a change in the pH of the culture medium from acidic to mildly alkaline [13]. These conditions reflect the physiology of in vivo infections: after oral ingestion, the bacteria pass through the acidic environment of the stomach to the mildly alkaline milieu of the small intestine, where they cause disease. Recently, the biochemical mechanisms utilized by a number of translocated effectors of Salmonella spp. to initiate invasion have been detailed (table I). Examples of translocated effectors which are encoded within SPI1 are SptP (Salmonella protein tyrosine phosphatase), SipA (Salmonella invasion protein; also called Ssp for Salmonella secreted protein), SipB, SipC, and AvrA (avirulence factor A). Many of these exert their effects on host cells in unique ways. SptP is a protein with phosphotyrosine phosphatase activity within a carboxy-terminal region and an aminoterminal region that bears resemblance to exotoxin S from Pseudomonas aeruginosa and YopE from Yersinia spp. [25]. Both domains of SptP disrupt the actin cytoskeleton when microinjected into epithelial cells, suggesting that the two domains cooperate to exert related functions upon translocation into host cells [26]. Invasion of epithelial cells in vitro does not require SptP, however mutants lacking this gene are impaired in their ability to colonize the spleen when coinfected in mice with wild-type S. typhimurium [25]. This competition assay reveals a subtle defect associated with SptP mutants, a common theme for other SPI1 translocated effector mutants. The Sip proteins (SipA, SipB, SipC, SipD) were first characterized based on their involvement in invasion of cultured epithelial cells [27, 28]. SipA binds directly to actin in the host cell, decreasing its critical concentration for polymerization and stabilizing actin filaments by inhibiting their depolymerization [29]. Binding of SipA to actin also activates T-plastin, causing an increase in the actin-bundling activity of this protein [30]. Moreover, sipA mutants do not induce actin rearrangements of the host cell at the site of bacterial contact [29]. These mutants are slightly decreased in invasion efficiency in that a difference is measurable early after infection, but not 1 h after initial infection. These findings suggest that SipA contributes to host cell actin cytoskeletal rearrangements that potentiate invasion. SipB and SipC are both directly involved in the translocation process, and are themselves delivered to the cytosol of the host cell. Upon infection of macrophages in vitro, SipB binds to the proapoptotic protease caspase-1 and rapidly induces apoptosis of the host cell [31]. Apoptosis may be important during the early stages of in vivo infection at Peyer’s patches, where resident macrophages intimately associate with M cells. Interestingly, S. dublin was found to induce apoptosis of epithelial cells but did so in a delayed manner [32]. How salmonella might differentially initiate apoptosis in epithelial cells and macrophages remains to be determined. SipC may also have an effector function in host cells. This is suggested by expression of receptor chimeras bearing the amino-terminus of this protein in epithelial cells, which block S. typhimurium invasion [33]. Thus, SipB and SipC appear to have dual funcMicrobes and Infection 2000, 145-156

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tion: as effectors and as translocators for other effector proteins of SPI1 (e.g., SptP, AvrA) Translocation of AvrA into host cells via the SPI1 encoded type III system has been demonstrated [34]. However, deletion of this gene does not have a measurable effect on in vitro invasion or the pathogenesis of S. typhimurium in mice, and its function remains unknown. AvrA displays homology with Yersinia YopJ [35] and the avirulence factor AvrRxv from Xanthomonas capestris pv. vesicatoria, the causative agent of leaf spot disease in pepper and tomato [36]. This homology provides the first example of targets of type III secretion systems that are conserved between plant and animal pathogenic bacteria. SopE is translocated by the SPI1 type III secretion system, yet it is encoded outside of centisome 63 within a cluster of genes from a cryptic P2-like prophage at 61 cs. It is present in only a subset of S. enterica serovars [37]. These observations suggest that sopE was probably horizontally acquired, and thus is an example of a Salmonella pathogenicity islet. Upon delivery into the host cell, SopE directly activates Cdc42 and Rac, two Rho family GTPases that are required for S. typhimurium invasion by functioning as a GTP/GDP exchange factor [38]. Activation of Cdc42 and Rac initiates a variety of downstream signal transduction cascades that lead to actin rearrangements and the production of pro-inflammatory cytokines [39]. As with SptP and SipA, deletion of SopE had little (though measurable) effect on in vitro invasion of epithelial cells [37]. Together, these studies highlight the multiple, and apparently redundant, mechanisms by which S. typhimurium engage host cell signalling pathways to effect a crucial host cell response for invasion, namely rearrangement of the actin cytoskeleton. Models of S. typhimurium invasion in vitro have demonstrated the profound ability of this pathogen to replicate within filamentous vacuoles following an initial lag period of 3–4 h after entry into epithelial cells. Experiments involving the uptake of a noninvasive invA mutant by coinfection with wild-type bacteria revealed that the SPI1 type III secretion system was absolutely required for replication to occur (O. Steele-Mortimer and B. Finlay, unpublished). Therefore, proteins that might allow the invA mutant to replicate could not be provided in trans by the wild-type strain. These findings suggest that SPI1 is required not only for invasion of nonphagocytic cells of the intestinal epithelium and apoptosis of macrophages, but possibly also the early and rapid replication within epithelial cells following invasion. Combined, these three functions would be expected to allow Salmonella spp. to establish a foothold in its niche within the gut of infected animals.

4. SPI2 In the past year, a great deal of progress has been made on the characterization of SPI2 (31 cs) [40, 41]. It contains more than 40 genes [42], including a two-component regulatory system and a type III secretion system (figure 1B). SPI2 forms an insertion of about 40 kb at the tRNAVal gene, and is divided into two segments, each of 149

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which may have been obtained in distinct horizontal transfer events [42]. The smaller 14.5-kb portion of SPI2 extends past ssrB to the 30.5 cs border of SPI2, containing a group of five ttr genes involved in tetrathionate reduction [43] and seven open reading frames (ORF) of unknown function [42]. This arm of SPI2 was probably acquired first due to its presence in the phylogenetically older species S. bongori. These genes do not appear to contribute significantly to systemic infections in the mice. The larger 25.3-kb portion of SPI2, located between tRNAVal gene at 31 cs of the S. typhimurium genome and ssrB of SPI2, may be a more recent insertion, because it is restricted to isolates of S. enterica [42]. It harbours genes important for virulence function. Four types of genes have been described in the 25.3-kb arm of SPI2, grouped into a least four operons (figure 1B). The genes are designated ssa (for genes encoding the type III secretion system apparatus), ssr (for secretion system regulators), ssc (for secretion system chaperones, and sse (for secretion system effectors) [44]. Four hours after invasion, S. typhimurium normally begins to replicate in mouse macrophage and human epithelial cell lines. However, most SPI2 mutants fail to replicate to the same extent as wild-type strains [45, 46]. Moreover, in orally infected mice, SPI2 mutants colonize the Peyer’s patches but are unable to spread to the mesenteric lymph nodes, liver or spleen [45]. Shea et al. [47] studied the growth kinetics of SPI2 mutants in different sites of the body during the course of an infection, using a nonreplicating plasmid to measure the relative rates of bacterial growth and killing. Their results suggest that the SPI2 type III secretion system is essential for the growth of bacteria, rather than for the survival against a host immune response. The type III secretion system of SPI2 is structurally and functionally distinct from that of SPI1. Crosscomplementation between type III systems of different species is possible. For example, S. typhimurium sipB can complement a Shigella flexneri ipaB mutant [48], and a S. typhimurium spaP mutant can be complemented by the corresponding spa of S. flexneri [49]. In contrast, many of the type III components of SPI2 do not compensate for mutations in the components of SPI1 within the same bacterium. Ssa genes are structurally most similar to Yersinia spp. ysc and lcr genes, and share weak but significant homology to EPEC proteins. There are at least two operons encoding structural components of the secretion apparatus: structural I, which includes spiCAB (ssaBCDE), and structural II, which includes ssaJKLMVN-U [45, 46]. The effector sse and ssc genes appear to be transcribed as a large nine-gene operon. This group of genes encodes proteins with homology to those secreted by type III secretion systems of EPEC, P. aeruginosa, and Yersinia spp. Two putative chaperones, SscA and SscB, have homology to the LcrH/IppI/SicA family of type III secretion chaperones. While it is apparent that SPI2 effector proteins are required for bacterial replication in macrophages, their exact molecular function is unclear. The predicted proteins of sseB, sseC, and sseD genes show weak, but significant, similarity to EPEC proteins EspA, EspD, and EspB, respectively. These EPEC proteins are required for the activation of epithelial signal transduction cascades 150

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that lead to host cell cytoskeletal rearrangements and the formation of pedestal-like structures on intestinal epithelial surfaces, to which the bacteria adhere. SseC and EspD are both similar to YopB of Y. enterocolitica, which is a pore-forming protein required for translocation of effector proteins by the Yersinia type III secretion system [50]. These similarities suggest that S. typhimurium delivers proteins into the SCV or through the vacuolar membrane into the host cytosol, which contribute to the survival and replication of S. typhimurium within host cells. Consistent with this prediction is the finding that SpiC is delivered to the cytosol of macrophages in a manner dependent on the Spi/Ssa type III secretion system. SpiC is thought to inhibit intracellular trafficking by inhibiting fusion of SCVs with lysosomes and endosomes, and may contribute to altered trafficking of the transferrin receptor [51]. Using transcriptional fusions to either green fluorescent protein or luciferase, SPI2 genes have been shown to be rapidly induced after entry into macrophages or epithelial cells and are continually expressed throughout infection [45, 52, 53]. Expression of structural genes is absolutely dependent on the two-component regulatory system encoded by ssrAB, both in vitro [54] and in vivo [45, 52]. SsrA (SpiR), a predicted 104-kDa protein, is the membrane-located sensor kinase [40], while SsrB is the transcriptional regulator sharing homology to a family of transcriptional activators including NarL and UvrY of E. coli, SirA of S. typhimurium, and BvgA of Bordetella pertussis [54]. Although work has been done to characterize the environmental cues that affect the expression of SPI2 genes, the exact signal integrated by the SsrAB system has yet to be determined. Treatment of S. typhimuriuminfected macrophages with bafilomycin, an inhibitor of the vacuolar proton ATPase which blocks endosomal pH acidification, abrogates induction of SPI2 genes. However, exposure of S. typhimurium to low pH in vitro does not induce expression of SPI2 genes [45, 54]. Therefore, other factors within the acidic vacuolar environment may induce the expression of SPI2 genes. Expression of SPI2 genes was monitored in vitro under various conditions using transcriptional fusions to luciferase, and antibodies against recombinant SPI2 proteins. The two main inducing conditions appear to be limiting concentrations of divalent cations and phosphate starvation [54]. Only low levels of SPI2 proteins are detected in bacteria lacking the twocomponent global transcriptional regulator PhoP/Q; however, a mutant with a constitutively active phoP allele does not produce constitutive SPI2 expression [54]. These results indicate that PhoP/Q, which is also present in nonpathogenic E. coli, acts as a modulator of SPI2 gene expression. The ssrAB local regulatory and the type III secretion genes were probably introduced together in a single horizontal genetic transfer event. The bacteria could then modulate SPI2 gene expression with the PhoP/Q system. This scenario is similar to the mgtCB genes of SPI3, which are also under the control of PhoP/Q (see below). It has been suggested that the type III secretion systems of SPI1 and SPI2 are inversely regulated [54]. This is an attractive hypothesis since in the host, salmonella must first invade M cells, where SPI1 expression is needed and Microbes and Infection 2000, 145-156

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then replicate inside macrophages, where SPI2 expression is needed. However, mutations in SPI2 genes encoding the type III secretion apparatus reduce the expression of the SPI1 genes sipC, prgK and hilA (encoding a transcriptional activator of SPI1 genes), suggesting that cross-talk exists between SPI2 and SPI1 [56]. Furthermore, the SPI1 type III secretion effector SipC can still be detected by immunofluorescence within cultured intestinal epithelial cells 2 hours after invasion, at which time PhoP/Q is active [55]. Thus, further study is required to sort out the complex gene regulation of SPI1 and SPI2.

5. SPI3 SPI3 (82 cs) is a 17-kb insertion at the selC tRNA locus, the same insertional site as that for EPEC and uropathogenic E. coli pathogenicity islands. Analysis of the GC content of SPI3 reveals a mosaic structure, and thus it appears to have evolved in a multistep process (figure 1B). SPI3 contains ten ORFs organized into six transcriptional units. Two of these genes, mgtCB, are required for intramacrophage survival and virulence in mice [57, 58]. These genes form an operon under the tight control of PhoP/Q. The encoded proteins allow S. typhimurium to transport magnesium at low Mg2+ conditions [57], although their function as Mg2+ transporters is still controversial [59, 60]. Unlike SPI1 and SPI2, SPI3 encodes proteins with no obvious functional relationship to each other [58]. Other SPI3-encoded proteins are MarT, which exhibits sequence similarity to the ToxR regulatory protein of Vibrio cholerae and MisL, which is similar to the AIDA-I adhesin of EPEC. Neither of these genes are required for virulence in BALB/c mice; however, they may be involved in other aspects of pathogenesis, such as chronic infection and host specificity [58].

6. SPI4 SPI4 (92 cs) is a 25-kb DNA insertion, flanked by ssb (a gene encoding the single stranded DNA binding protein), and soxSR (encoding superoxide response regulatory genes) (figure 1B). There is also a putative tRNA-like gene and transcriptional terminator structure at the boundaries of SPI4 [61]. Like SPI3, SPI4 also has a mosaic structure. The ORFs have a lower GC content compared to the Salmonella chromosome, while the intergenic regions have a higher GC content. SPI4 has eighteen putative ORFs, and from sequence analysis, may encode a type I secretion system that mediates toxin secretion, much like E. coli hemolysin secretion. A number of toxins have been isolated from bacteria that induce apoptosis in several immune cells, including leukotoxin from Pasturella haemolytica and adenylate cyclase-hemolysin from B. pertussis. Since S. typhimurium induces apoptosis of infected macrophages, it has been speculated that SPI4 is involved in secretion of a cytotoxin [61]. Consistent with this hypothesis, a number of groups have reported cytotoxicity induced on macrophages by several Salmonella species [62, 63, 64, 65]. A SPI4-dependent mechanism of Microbes and Infection 2000, 145-156

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inducing apoptosis may act in concert with the binding of SipB to caspase-1 (see earlier SPI1 section and [31]). Finally, a locus within SPI4 is required for intramacrophage survival [66]; however, the main function of SPI4 remains to be determined.

7. SPI5 SPI5 is located at 25 cs on the S.dublin chromosome and has been identified in a wide variety of S. enterica spp., including S. typhimurium [67, 68], although it appears to be absent in other enteropathogenic bacteria. This insertion is flanked by serT and copS/copR and its overall GC content is approximately 43.6%. The role of SPI5 appears mainly associated with enteropathogenesis: mutations in either S. dublin pipD (pathogenicity island encoded protein), sopB (Salmonella outer protein), pipB or pipA (figure 1B) has minimal effect on systemic infection in mice, but displays markedly attenuated secretory responses in a bovine ligated ileal loop model of enteritis [67]. Interestingly, a sopB and sopD double mutation (sopD is not located within SPI5) had an additive effect on the inhibition of secretion in this assay, suggesting that these virulence factors act in concert [69]. SopB is translocated into the host cytosol, where it mediates inflammation and fluid secretion in intestinal mucosa [70]. Furthermore, translocation of SopB occurs in a SipB-dependent manner, suggesting that the SPI1 type III secretion system mediates its delivery to host cells. SopB is an inositol phosphate phosphatase whose activity yields inositol 1,4,5,6-tetrakisphosphate, a signalling molecule which further enhances the chloride secretion that may be associated with fluid influx [71]. In addition, SopB hydrolyzes phosphatidylinositol 3,4,5-trisphosphate, an inhibitor of Ca2+-dependent chloride secretion in epithelial cells. SopB contains two small regions of sequence homology to mammalian inositol phosphate 4-phosphatases, one of which is thought to be the putative catalytic site of these enzymes. Mutation of sopB at a highly conserved cysteine residue in the putative catalytic site results in decreased fluid secretion in the bovine ileal loop model of enteritis [71]. Thus, Salmonella spp. have the ability to engage both lipid and protein signalling pathways in host cells. SigD (Salmonella invasion gene), and SigE are S. typhimurium homologues of the S. dublin SopB and PipC, respectively. They were initially identified in a genetic screen for novel invasion loci using a SPI1 mutant strain [68]. However, the effect of a sigD mutation on invasion appears to depend on where the mutation is introduced, whereas the reported sopB mutants display normal invasiveness in vitro. It is thought that SigD and SopB have similar roles in vivo [53]. The downstream gene pipB in S. dublin appears to be required for enteropathogenesis. However, a mutation in the S. typhimurium pipB homologue results in a slight reduction in virulence when administered intravenously, suggesting that PipB has a role outside of enteropathogenesis in the murine typhoid model. Clearly, further study of pipB, and sopB /sigD will be required to define their individual roles. 151

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8. Salmonella virulence plasmid Many Salmonella serovars harbour virulence plasmids important for systemic infection of experimental animals after oral inoculation [72, 73]. The Salmonella virulence plasmid varies in size, depending on the serovar: 95 kb for S. typhimurium; 60 kb for S. enteritidis; 80 kb for S. dublin; and ranging from 50 to 110 kb for S. choleraesuis [74]). Moreover, a highly conserved 8-kb operon of five genes, designated spvRABCD (for Salmonella plasmid virulence) is present in all Salmonella serotypes examined, and can restore virulence to plasmid-cured strains in a mouse model (reviewed in [77]). This region may have been horizontally acquired, since it is located adjacent to an insertional element and the GC content is lower (46%) than that of the overall S. typhimurium chromosome. spvB is rapidly induced inside cultured mammalian cells [78]. Expression is not affected by alkalinization of the SCV, but is dependent on SpvR, a member of the LysR family of transcriptional activators. SpvR is controlled by different regulatory factors depending on the growth conditions, such as nutrient availability and growth phase [79]. SpvA is negative regulator of the operon [80], and SpvBCD may be membrane-associated proteins, although their activities remain obscure. The spv region appears to promote the survival and rapid growth of salmonella in the host, thereby increasing virulence. However, several groups have studied the role of spv in vitro in macrophages, and found no relationship between spv gene expression and intracellular survival [65, 75, 76]. Therefore, the role of the Salmonella virulence plasmid is more complex than was first thought and awaits further study. Gulig et al. [81] examined the effect of spv region on the growth of S. typhimurium in vivo within different populations of mouse host cells, including macrophages, neutrophils, and lymphocytes. A genetic marker to measure the relative numbers of bacterial cell divisions in mice was used in mixed infections of S. typhimurium, with or without the virulence plasmid. Depletion of macrophages, but not neutrophils or lymphocytes, rendered the two strains equally virulent. Therefore, of the cell types examined, only macrophages appear to be relevant in the virulence mediated by the plasmid. Another study [82] reveals a complex interplay between plasmid virulence genes in Salmonella, host macrophages and neutrophils. BALB/c mice have a point mutation in the macrophageexpressed gene, Nramp1, that renders them susceptible to Salmonella infection. Mice with normal macrophages (i.e., with a functional Nramp1) need polymorphonuclear leucocytes (PMNs) to defend against S. dublin that carries the virulence plasmid, but not against plasmid-cured S. dublin. Thus, it appears that the virulence mechanisms conferred by the virulence plasmid allow salmonella to survive in macrophages but does not protect them from PMNs. A possible role of the spv genes in the apoptosis of macrophages remains unclear. Guilloteau et al. [65] analysed the interaction of wild-type and plasmid-cured strains of several Salmonella serovars in murine and bovine macrophages after infection in vivo or in vitro. The presence of the virulence plasmid resulted in increased 152

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lytic activity of S. typhimurium, S. dublin, and S. choleraesuis for both resident and activated mouse peritoneal macrophages. Lysis was not mediated by the spvRABCD genes, suggesting the existence of uncharacterized plasmid genes involved in macrophage lysis. In contrast, a recent study has reported that the Salmonella-induced cytopathology in human monocyte-derived macrophages does require the spv genes, since an spvR mutation abolished the cytopathic effect [83].

9. Summary/future directions From this discussion of SPIs, it is sometimes unclear how certain DNA regions are designated as pathogenicity islands. Small DNA regions (often single genes) which are horizontally acquired are numerous, and their distinction from pathogenicity islands is in some cases arbitrary. For example, SPI3 has a mosaic structure, suggesting that acquisition of the parts involved distinct transfer events. Moreover, within SPI3, only the genes mgtCB are considered virulence genes and were likely acquired independently of other areas of SPI3. Thus, mgtCB may be considered more of a pathogenicity islet like sifA, the gene critical for production of Salmonella-induced filaments [84]. Currently, a pathogenicity island is considered to be a stable insertion of a large region of DNA, containing virulence genes, into a site in the bacterial chromosome. This region usually contains complex virulence determinants, which appear to be acquired in a single step. For example, the distribution and the GC contents of genes encoding the type III secretion systems on SPI1 and SPI2 suggest that both DNA regions were acquired in single events. However, this raises the issue of the size of these pathogenicity islands. For instance, should the genes involved in tetrathionate reduction, which were obtained in a separate horizontal transfer event from the SPI2 type III secretion genes, really be considered part of SPI2? Similarly, within SPI1, the invH gene located at one border shows a different distribution among Salmonella serotypes than the genes encoding the SPI1 type III secretion system apparatus [85]. In the case of SPI4 and SPI5, this information (which appears to be relevant for categorizing the corresponding DNA regions as pathogenicity islands) is not yet available. Thus, a more critical discussion of what really constitutes a pathogenicity island is warranted in the future. It is striking how salmonella has adapted, apparently in large genetic leaps, into a multitude of species and serovars that are capable of infecting a diverse range of animals from reptiles to mammals. Animal models using host-specific salmonella have allowed the study of pathogenicity in a more accurate way, and reveal that SPIs play a major role in host range and pathology of infection. SPI1 and SPI5 appear to have their virulence function restricted to the gut, while SPI2, 3, and 4 and the virulence plasmid seem to have adapted Salmonella spp. for growth in macrophages. Evidence for cross-talk between the SPIs suggests the existence of complex integrated regulatory networks to provide various levels of regulation: SPI2 influences SPI1; and PhoP/Q modulates expression of Microbes and Infection 2000, 145-156

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genes within SPI1, SPI2, SPI3, and probably others. Moreover, effector proteins encoded outside of SPI1, such as SopE and SopB are translocated by the SPI1 type III secretion system. Still, much remains unclear about the regulation of SPI genes and how the virulence factors in SPIs exert their functions during the course of an infection. The five SPIs so far described are not present in all species of Salmonella, which may contribute to the varying host specificity. Consistent with this idea, a number of acquired genetic elements were identified, outside of the SPIs, that have unusual base composition, are associated with mobile elements and are required for full virulence [86]. These properties are hallmarks of pathogenicity islands. Some of these regions encode molecules predicted to be involved in adhesion and invasion, and distinguish broad host range from host-adapted Salmonella serovars. Moreover, there is evidence that S. typhi, but not S. typhimurium, may use the cystic fibrosis transmembrane conductance regulator to enter epithelial cells, suggesting that the ability to invade epithelial cells is critical for causing human typhoid fever [87]. The advent of genome sequencing will make it possible to compare the genomes of multiple Salmonella spp. such as S. typhi and S. typhimurium. These studies will hopefully shed light on the genes required for infection of the specific hosts of each species, and hopefully further define the determinants of gastroenteritis and systemic disease. At the very least, they will identify the unique sequences in salmonella that are not found in their close relatives, E. coli and Shigella spp. The continued search for the host targets of SPI effectors may also open up a new class of therapeutic drugs in the treatment of antibiotic-resistant Salmonella infections. It is clear that SPIs play a key role in Salmonella pathogenesis. Defining these genetic elements, and elucidating their functions, will provide a much-improved understanding of how Salmonellae cause disease.

Acknowledgments We thank members of the Finlay laboratory and José Luis Puente for critical reading of the manuscript, and Olivia Steele-Mortimer for help with the figures. S.L.M is supported by a fellowship from the Medical Research Council of Canada, and has an honourary fellowship from the Alberta Heritage Foundation for Medical Research. J.H.B. is supported by a fellowship from the Natural Sciences and Engineering Research Council of Canada and is an honourary Izaac Walter Killam fellow. Work in B.B.F.’s laboratory is supported by the Medical Research Council of Canada. B.B.F. is a Howard Hughes International Scholar and an M.R.C. scientist.

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