1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Pathogenicity islands (PAIs) are large DNA segments in the genomes of bacterial pathogens that encode virulence factors. Five PAIs have been identified in the Gram-negative bacterium Salmonella enterica. Two of these PAIs, Salmonella pathogenicity island (SPI)-1 and SPI-2, encode type III secretion systems (TTSS), which are essential virulence determinants. These ‘molecular syringes’ inject effectors directly into the host cell, whereupon they manipulate host cell functions. These effectors are either encoded with their respective TTSS or scattered elsewhere on the Salmonella chromosome. Importantly, SPI-1 and SPI-2 are expressed under distinct environmental conditions: SPI-1 is induced upon initial contact with the host cell, whereas SPI-2 is induced intracellularly. Here, we demonstrate that a single PAI, in this case SPI-5, can encode effectors that are induced by distinct regulatory cues and targeted to different TTSS. SPI-5 encodes the SPI-1 TTSS translocated effector, SigD/SopB. In contrast, we report that the adjacently encoded effector PipB is part of the SPI-2 regulon. PipB is translocated by the SPI-2 TTSS to the Salmonella-containing vacuole and Salmonella-induced filaments. We also show that regions of SPI-5 are not conserved in all Salmonella spp. Although sigD/sopB is present in all Salmonella spp., pipB is not found in Salmonella bongori, which also lacks a functional SPI-2 TTSS. Thus, we demonstrate a functional and regulatory cross-talk between three chromosomal PAIs, SPI-1, SPI-2 and SPI-5, which has significant implications for the evolution and role of PAIs in bacterial pathogenesis.


  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Horizontal gene transfer is an important source of bacterial evolution. The evolution of pathogenic microbes has been impacted particularly by the acquisition of large genomic elements called pathogenicity islands (PAIs). PAIs encode one or more virulence factors, often including type III secretion systems (TTSS) (Hacker and Kaper, 2000). The genetic acquisition of TTSS was a major evolutionary leap for Gram-negative bacterial pathogens. TTSS allow animal and plant pathogens to inject their own proteins, termed effectors, directly into host cells and, subsequently, to modulate specific host cellular functions (Hueck, 1998). These ‘molecular syringes’ and their secreted effectors are essential virulence determinants. Salmonella enterica possess two TTSS. These are chromosomally encoded in large PAIs and are designated Salmonella pathogenicity island (SPI)-1 and SPI-2 (Galan, 2001). Both SPI-1 and SPI-2 encode translocated effector proteins and regulatory proteins, in addition to the molecular machinery required for TTSS. Unlike most other Gram-negative pathogens with TTSS, Salmonella also translocate effectors that are not encoded within SPI-1 or SPI-2 (Galan, 2001). The two TTSS of Salmonella function at disparate stages of the Salmonella infection process, yet both are essential for complete virulence (Galan and Curtiss, 1989; Ochman et al., 1996; Shea et al., 1996). SPI-1 is required for Salmonella to breach the host cell membrane. For example, several SPI-1 translocated effectors act in conjunction to induce, and then dampen, membrane ruffling, which results in Salmonella invasion of epithelial cells (Hardt et al., 1998; Fu and Galan, 1999; Hayward and Koronakis, 1999; Zhou et al., 1999a; 2001; Mirold et al., 2001). After bacterial invasion, Salmonella reside within an acidified vacuole called the Salmonella-containing vacuole (SCV). The SPI-2 TTSS is presumed to translocate effectors across the SCV membrane, although very few SPI-2 translocated proteins have been identified, and even fewer have been characterized. SPI-2 is induced after bacterial internalization and is essential for the ability of Salmonella to survive and replicate within this vacuole in host cells, particularly phagocytic cells such as macrophages (Cirillo et al., 1998; Hensel et al., 1998). The evolution of S. enterica to cause systemic infection in the host probably results from the acquisition of the SPI-2 TTSS (Baumler et al., 1998).

SPI-5 is a PAI located at 24 centisomes on the Salmonella chromosome. In S. enterica serotype Dublin (S. Dublin), SPI-5 encodes at least five genes, pipD, sigD/sopB, sigE, pipB and pipA, all of which contribute to enteropathogenesis as assessed in a calf model of infection (Wood et al., 1998). Only one effector protein from SPI-5, SigD/SopB, has been characterized. SigD/SopB is an inositol polyphosphate phosphatase (Norris et al., 1998) that plays a multifaceted role in Salmonella pathogenesis, including activation of the mammalian protooncogene Akt (Steele-Mortimer et al., 2000) and promotion of host cell membrane ruffling and invasion (Hong and Miller, 1998; Mirold et al., 2001; Zhou et al., 2001). SigD/SopB is co-ordinately regulated with SPI-1 genes (Darwin and Miller, 2001) and is secreted by the SPI-1 TTSS, which requires a molecular chaperone SigE (Darwin et al., 2001). Recent work in our laboratory (Pfeifer et al., 1999) identified two genes within SPI-5, sigD/sopB and pipB, as being induced inside host cells during Salmonella infection. Comparison of both the kinetics and the intensity of intracellular sigD/sopB and pipB expression suggested that these two genes responded to different regulatory cues. In this paper, we show that, unlike SigD/SopB, PipB is expressed under SPI-2-inducing conditions. Furthermore, we identify PipB to be a SPI-2 TTSS translocated protein that localizes to SPI-2-induced structures in host cells called Salmonella-induced filaments (Sifs). We thus introduce the new concept that effectors encoded within a single PAI can be induced by dissimilar environmental signals and translocated by distinct TTSS. For the acquisition of new genetic information via lateral transfer to be advantageous to a pathogen, such genes need to be functionally co-ordinated with others in the bacterial genome. A fine example is presented here, in which the differential targeting of these two effectors results, at least in part, from co-ordinate expression with their dedicated TTSS. The regulatory and functional cross-talk between three chromosomal PAIs, SPI-1, SPI-2 and SPI-5, is a remarkable illustration of how chromosomal PAIs have co-evolved to generate an integrated network of virulence functions in pathogenic bacteria.


  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

SPI-5 genes have a differential expression pattern

To investigate the expression profile of four genes within SPI-5, whose genetic organization is depicted in Fig. 1A, reverse transcription–polymerase chain reaction (RT–PCR) analysis was performed using RNA extracted from bacteria grown under two different conditions to induce SPI-1 and SPI-2 expression. SPI-1 gene expression is induced in rich culture broth at the mid-log phase of growth (Chen et al., 1996; Lundberg et al., 1999). In contrast, SPI-2 genes are poorly expressed in rich culture media, but are induced in minimal media with low concentrations of Mg2+ or Ca2+, conditions that probably mimic those inside the SCV (Deiwick et al., 1999). Expression of the first gene within SPI-5, pipD, was unchanged in SPI-1 and SPI-2-inducing media (Fig. 1B). As expected, expression of sigD/sopB, which encodes a SPI-1 TTSS secreted effector (Hong and Miller, 1998), was markedly increased in SPI-1-inducing media (Fig. 1B). Surprisingly, the expression profiles of pipB and pipA were very different from that of sigD. Both these genes were preferentially expressed in SPI-2-inducing media (Fig. 1B).


Figure 1. Genetic analysis of SPI-5.

A. Genetic organization of SPI-5 (adapted from Wood et al., 1998).

B. RT–PCR analysis of pipD, sigD/sopB, pipB and pipA indicates differential expression of these genes. Total RNA was isolated from S. Typhimurium grown in SPI-1 (1)- or SPI-2 (2)-inducing conditions as described in Experimental procedures. The comparative abundance of four genes from SPI-5 was then analysed by RT–PCR.

C. Northern analysis of sigD/sopB and pipB shows a distinct expression profile for each gene under SPI-1- and SPI-2-inducing conditions. Total RNA was isolated from S. Typhimurium as in (B). Molecular markers (kb) are shown on the lefthand side.

D. Mapping the start site of the pipB transcript. 5′ RACE analysis of total RNA isolated from bacteria grown under SPI-2-inducing conditions identified the start site of the pipB transcript. Two PCR products were obtained, and both were cloned into pGEM T-Easy. Arrowheads mark each transcript start as determined by sequence analysis. A putative ribosome binding site (RBS) and potential σ70-dependent –10 hexamers are underlined. No –35 hexamers were obvious, as has been described previously for some other Salmonella gene transcripts (Darwin and Miller, 2001).

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Northern analysis of total RNA extracted from S. enterica serovar Typhimurium (S. Typhimurium) grown under SPI-1- or SPI-2-inducing conditions confirmed the RT–PCR results for sigD/sopB and pipB. Using a sigD/sopB probe, a transcript of ≈ 2.5 kb was abundant in SPI-1-inducing conditions only (Fig. 1C). This is the predicted size of the sigDE operon. Conversely, for pipB, an ≈ 1.0 kb transcript was detected predominantly in SPI-2-inducing conditions (Fig. 1C), which is the predicted size of the pipB open reading frame (ORF). This is in contrast to the findings Wood et al. (1998), who reported a polycistronic transcript for sigD/sopB, sigE, pipB and pipA, although the bacterial growth conditions for their analysis were not reported.

Both RT–PCR and Northern analysis demonstrated a very different expression profile for the adjacent genes pipB and sigD/sopB. This suggested that these two genes are under different regulatory controls. Darwin and Miller (2001) have recently mapped the sigD/sopB promoter start site to 57–58 bp upstream of the sigD/sopB ATG start codon. We wished to identify the trans-criptional start site for pipB. Using total RNA isolated from SPI-2-inducing growth conditions, 5′ random amplification of cDNA ends (RACE) analysis of pipB amplified two PCR products of different sizes (results not shown). After cloning into pGEM T-Easy, five clones from each PCR product were sequenced. This yielded two transcript start sites for pipB– one species initiating 49–50 bp upstream of the ATG start codon and a longer transcript mapping to –184 bp (Fig. 1D). Thus, the pipB promoter is located in the intergenic region between sigE and pipB and is induced under SPI-2 growth conditions. Taken together, this demonstrates that pipB is transcribed independently of the sigD/sopB promoter.

PipB is induced under SPI-2 growth conditions and is dependent on SsrA/SsrB

We next investigated the expression of PipB in different growth media using an epitope-tagged PipB. Bacteria expressing haemagglutinin (HA)-tagged PipB under the control of the pipB promoter [ΔpipB (pACB C-2HA)] were grown in SPI-1- or SPI-2-inducing media as described, and proteins were subject to Western analysis. In concordance with the transcript analysis results, SigD/SopB and PipB had very different expression profiles (Fig. 2A and B). Although SigD was expressed under SPI-1-inducing conditions, PipB expression was greatly induced by growth in minimal media. PipB expression levels were comparable for wild-type bacteria and an SPI-2 TTSS mutant, ΔssaR (Fig. 2B). SPI-2 gene expression is absolutely dependent upon the SPI-2-encoded two-component regulatory system SsrA/SsrB (Cirillo et al., 1998; Deiwick et al., 1999). PipB expression was dramatically reduced in a ssrB::kan mutant (Fig. 2B), demonstrating that PipB is part of the SsrB regulon. Indeed, the expression profile of PipB was comparable with that of SseB (Fig. 2B), an SPI-2-encoded protein that is secreted by the SPI-2 TTSS (Beuzon et al., 1999).


Figure 2. PipB is induced by SPI-2 growth conditions.

A. SigD/SopB is induced by SPI-1-inducing growth conditions. Bacteria [ΔsigD (pACDE C-myc)] were grown as described in Experimental procedures, and proteins corresponding to an equal number of bacteria were loaded in each lane. Expression of SigD tagged with a C-terminal myc epitope and DnaK were monitored by Western blot analysis.

B. PipB expression is induced by SPI-2 growth conditions and is dependent on the SPI-2 transcriptional activator SsrB. Bacteria [ΔpipB (pACB C-2HA) and ΔpipB ssrB::kan (pACB C-2HA)] were grown as described in Experimental procedures. HA-tagged PipB and DnaK were detected by Western analysis. The lower blot shows a comparable expression pattern for SseB, an SPI-2-encoded protein.

C. PipB expression is induced intracellularly. Western analysis of HA-tagged PipB expression from ΔpipB (pACB C-2HA) infected HeLa (top) or RAW264.7 (bottom) cells as per Experimental procedures. Samples were standardized to an equal number of bacteria for each time point. Intracellular induction of HA-tagged PipB expression is not detected for an SsrB mutant (ssrB::kan).

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Genes that comprise the SPI-2 regulon are specifically induced within host cells (Cirillo et al., 1998). To examine the kinetics of PipB expression in intracellular bacteria, we infected both phagocytic and non-phagocytic cells with the ΔpipB (pACB C-2HA) strain and measured HA-tagged PipB by Western analysis. Over the time course, samples were normalized to equivalent bacterial colony-forming units (cfu). PipB was not detected until 3 h after bacterial infection of both HeLa epithelial and RAW 264.7 macrophage-like cells (Fig. 2C). Thereafter, relative PipB expression increased up to 21 h post infection (p.i.) (Fig. 2C). Thus, PipB expression is induced after bacterial entry into mammalian cells, with kinetics expected for a SPI-2-regulated gene. Indeed, we have shown previously using translational fusions that the kinetics of intracellular expression are comparable for PipB and the SPI-2-encoded SsaR (Pfeifer et al., 1999). Of note, this pattern of expression was markedly different from that of SopB/SigD (Pfeifer et al., 1999). The intracellular expression of PipB was absolutely dependent on SsrB in both RAW 264.7 macrophage-like cells (Fig. 2C) and HeLa epithelial cells (not shown). The lack of PipB-HA signal for the ΔpipB ssrB::kan strain was not because of insufficient bacterial numbers resulting from a replication defect, as equivalent bacterial cfu were loaded for the ΔpipB and ΔpipB ssrB::kan time courses. Taken together, this demonstrates that PipB expression is co-ordinately regulated with that of SPI-2-encoded genes.

PipB–CyaA fusions are translocated into the host cell

In order to examine whether PipB was translocated into host cells, fusions of the adenylate cyclase toxin (CyaA) from Bordetella pertussis to various N-terminal fragments of PipB were created. CyaA fusions have been used in other studies to monitor successfully the translocation of bacterial effectors (Schesser et al., 1996; Jones et al., 1998; Miao et al., 1999; Miao and Miller, 2000). In this system, if the CyaA fusion is translocated into the host cell cytoplasm, CyaA converts ATP into cAMP in the presence of calmodulin, a cytoplasmic protein. The resulting increases in cAMP can be measured by immunoassay. Using such approaches for other Salmonella effectors, regions necessary for translocation have been localized to the N-terminal regions of these proteins. For instance, the amino-terminal 202 residues of SopD are sufficient for the translocation of a SopD–CyaA fusion into host cells in a Sip-dependent manner (Jones et al., 1998). Miao and Miller (2000) have recently reported that 143 N-terminal residues of SspH2 is the minimal translocation signal for SspH2–CyaA fusions. We thus constructed CyaA fusions to different N-terminal regions of PipB, ranging from the first 90 to 210 amino acids. When we infected HeLa cells with wild-type S. Typhimurium for 5.5 h, only a small amount of cAMP could be detected in cell extracts (Fig. 3). No detectable increases in cAMP resulted from S. Typhimurium expressing CyaA fusions to the amino-terminal 90 or 120 amino acids of PipB (Fig. 3). Fusion of CyaA to the 150 N-terminal residues of PipB was sufficient for a measurable increase in host cell cAMP levels (Fig. 3). However, fusions to 180 or more N-terminal residues of PipB resulted in substantially greater increases in intracellular cAMP compared with 150 N-terminal residues of PipB. Taken together, these results demonstrate that PipB is translocated into host cells and that the TTSS recognition signal lies within the N-terminal 180 residues of PipB.


Figure 3. N-terminal PipB–CyaA fusions are translocated into host cells. HeLa cells were infected for 5.5 h with S. Typhimurium alone (–) or S. Typhimurium expressing various PipB–CyaA fusions from the native pipB promoter. For example, N90 is CyaA fused to the N-terminal 90 amino acid residues of PipB. cAMP levels in HeLa cell extracts were monitored by enzyme immunoassay as described in Experimental procedures. Values were normalized for protein content and represent the mean ± SD from three separate experiments.

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PipB is translocated by the SPI-2 TTSS to SPI-2-induced host cell structures

As CyaA fusion data indicated that PipB was delivered into host cells, we used immunofluorescence microscopy analysis of HeLa cells infected with Salmonella expressing HA-tagged PipB to detect the intracellular location of translocated PipB. At early times of infection (<6 h), PipB was not detectable by immunofluorescence. After 8 h of infection, PipB was localized around intracellular bacteria and also to filamentous structures resembling Salmonella-induced filaments (Sifs) (Fig. 4A). Sifs interconnect with the SCV, the compartment in which intracellular Salmonella reside. They are formed in an SPI-2-dependent manner in epithelial cells (Beuzon et al., 2000; Brumell et al., 2001) coincident with the onset of bacterial replication (Garcia-del Portillo et al., 1993). Figure 4B demonstrates that PipB is indeed translocated to Sifs, as PipB staining co-localized with LAMP-2, a lysosomal glycoprotein that is recruited to these Salmonella-induced structures (Stein et al., 1996). The SCV staining by PipB at 8 h p.i. was often in a ‘cloud-like’ or ‘halo’ pattern (see arrowhead in Fig. 4A), rather than in tight association with the bacteria. At later time points, the Sif network was more extensive, as shown by the marked PipB localization pattern in Fig. 4A. PipB also remained associated with the SCV. This localization was not specific to epithelial cells, as HA-tagged PipB also localized to the SCV and tubular structures resembling Sifs in phagocytic cells (RAW 264.7 macrophage-like cells; not shown).


Figure 4. PipB is a Salmonella-translocated protein.

A. PipB is translocated to the SCV and Sifs. HeLa cells were infected with S. Typhimurium ΔpipB (pACB C-2HA), expressing HA-tagged PipB, for 8 h or 21 h. Cells were fixed, permeabilized and immunostained for Salmonella LPS (left) and HA (middle). An overlay of the two images is shown on the right. Bar = 10 μm; arrowhead indicates a ‘halo’-like PipB staining pattern.

B. PipB staining co-localizes with LAMP-2. HeLa cells were infected for 8 h and processed as in (A) except that immunostaining was for LAMP-2 (left) and HA (middle).

C. PipB is translocated by the SPI-2 TTSS. HeLa cells were infected for 21 h with a SPI-1 (invA::kan) or SPI-2 (ΔssaR) TTSS mutant. Immunostaining was for Salmonella LPS (left) and HA-tagged PipB (middle). An overlay is presented on the right.

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To determine whether PipB translocation was via the SPI-1 or SPI-2 TTSS, we constructed ΔpipB invA::kan (pACB C-2HA) and ΔpipBΔssaR (pACB C-2HA) mutants. Both InvA and SsaR are structural components of the SPI-1 and SPI-2 TTSS respectively (Galan et al., 1992; Hensel et al., 1997). To compensate for the invasion deficiency of the SPI-1 mutant, HeLa cells were incubated with 100-fold more bacteria for 60 min instead of 10 min as per the normal infection conditions. Despite the lack of a functional SPI-1 TTSS, PipB was still localized to SCV and Sifs at both 8 h (not shown) and 21 h p.i. (Fig. 4C). Although SPI-2 mutants fail to induce Sif formation (Guy et al., 2000; Beuzon et al., 2000; Brumell et al., 2001), they remain enclosed within a vacuole (Beuzon et al., 2000). However, translocation of PipB to the SCV was not detectable using the SPI-2 TTSS mutant (Fig. 4C). This was not the result of decreased synthesis of PipB, as intracellular levels of PipB were comparable between wild-type bacteria and a ΔssaR mutant under SPI-2-inducing growth conditions (Fig. 2B). This demonstrates that the bacterial translocation of PipB to SCV and Sifs requires a functional SPI-2 TTSS.

As we have shown that PipB is translocated to both the SCV and Sifs, we assessed whether PipB was involved in the formation or maintenance of these Salmonella-induced structures. HeLa cells were infected for 8 h with either wild-type S. Typhimurium or the ΔpipB mutant, fixed and stained with antibodies against lipopolysaccharide (LPS) and LAMP-2. In three separate experiments, counting at least 100 infected cells for each experiment, 62 ± 2% of HeLa cells infected with wild-type Salmonella were positive for Sifs. This was comparable with 60 ± 5% Sif-positive cells for the ΔpipB mutant after 8 h of infection. Furthermore, as judged by LAMP-2 staining, the Sifs affected by wild-type Salmonella and the ΔpipB mutant are morphologically indistinguishable (not shown). Similarly, there was no difference in the association of LAMP-2 with SCV surrounding wild-type Salmonella and the ΔpipB mutant. At 8 h p.i., LAMP-2 was associated with 92 ± 5% of wild-type bacteria and 94 ± 5% of ΔpipB bacteria. Collectively, these data demonstrate that PipB is not required for the formation or maintenance of either the SCV or Sifs.

PipB is not required for intracellular survival or virulence

SPI-2 plays an important role in intracellular parasitism. As such, many SPI-2 mutants are defective for intracellular survival and replication in phagocytic cells (Cirillo et al., 1998; Hensel et al., 1998). As we have determined PipB to be part of the SPI-2 regulon, we assayed the ability of a ΔpipB mutant to survive and replicate within RAW264.7 cells using the gentamicin protection assay. Opsonized bacteria were used to infect cells to minimize Salmonella-induced cytotoxicity (Cirillo et al., 1998). As shown in Fig. 5A, the ΔpipB mutant was not impaired in intracellular growth in phagocytic cells compared with wild-type bacteria. In contrast, the SPI-2 mutant ΔssaR showed a characteristic defect in intracellular survival and replication (Fig. 5A). Hence, PipB is not required for intracellular replication in phagocytic cells.


Figure 5. PipB is not required for intracellular replication in host cells or virulence in mice.

A. Intracellular survival and replication of S. Typhimurium SL1344 (wt), a ΔpipB mutant (ΔpipB) and an SPI-2 mutant (ΔssaR) in RAW264.7 cells. After opsonization and internalization, gentamicin-protected bacteria were enumerated at 2 h and 24 h p.i. The values shown are the fold increase in the number of intracellular bacteria over this time frame. Values represent the mean ± SD from three independent experiments.

B. PipA, and not PipB, is required for virulence in mice. Survival of BALB/c mice was recorded over time after oral inoculation (five mice per group) with ≈ 1 × 104 cfu of S. Typhimurium SL1344 (open squares), ΔpipB (closed circles), ΔpipA (closed triangles) or E12A2 (pipB::luxABtet) (Pfeifer et al., 1999) (closed squares). Data are from a representative experiment.

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We have shown previously that an insertional mutation in pipB (pipB::luxABtet) reduced bacterial virulence in the mouse model of infection (Pfeifer et al., 1999). Owing to the possibility of this mutation having polar effects on the downstream gene pipA, we compared the virulence in mice of a non-polar gene deletion mutant ΔpipB and a gene deletion mutant ΔpipA with the pipB insertional mutant (E12A2). Groups of five mice were orally inoculated with 1 × 104 cfu of S. Typhimurium SL1344, ΔpipB, ΔpipA or the pipB insertional mutant (E12A2). Survival curves from a representative experiment are shown in Fig. 5B. By day 10 p.i., all the mice infected with either S. Typhimurium wild type or the ΔpipB mutant had died. In contrast, only three of the five mice infected with ΔpipA or E12A2 had died over the same interval. At day 21 p.i., one mouse remained for both the ΔpipA and the E12A2 infection groups. These mice were sacrificed, and the small bowel, mesenteric lymph nodes and spleen were recovered and homogenized. No cfu were detectable from these organs when plated on LB agar containing 100 μg ml–1 streptomycin. Thus, infection with either E12A2 or ΔpipA resulted in decreased mortality compared with wild-type S. Typhimurium. This demonstrates that our initial observation of reduced virulence in a mouse model for the pipB insertional mutant E12A2 resulted from polar effects on the downstream gene pipA. This implies that pipBA is transcribed as an operon. In conclusion, using this method to assess virulence, PipA but not PipB contributes overtly to the development of systemic disease in mice.

pipB is not present in all Salmonella strains

Phylogenetic analyses have shown that the SPI-1 TTSS is present in all contemporary Salmonella lineages (Boyd et al., 1997) (Table 1), but genes encoding for the SPI-2 TTSS are only found in S. enterica lineages, not in Salmonella bongori (Ochman and Groisman, 1996; Hensel et al., 1999) (Table 1). With our observation that SigD/SopB and PipB are translocated by different TTSS, we examined the distribution of pipB in Salmonella spp. Mirold et al. (2001) have recently shown that sigD/sopB is present in every isolate of the Salmonella reference collection C (SARC). Southern hybridization analysis using a pipB probe showed that this gene is present in all 72 S. enterica subspecies I strains of the Salmonella reference collection B (SARB; results not shown). We subsequently analysed the distribution of pipB among strains of SARC. This collection includes strains from the seven S. enterica subspecies and two S. bongori serotypes. Under low-stringency hybridization conditions, only strains belonging to S. enterica subspecies I (SARC1 and SARC2) and subspecies IIIa (SARC5 and SARC6) yielded signals with the pipB probe, but their intensity was weak (results not shown), presumably because of the small probe size. We performed further analyses using a probe for the coding sequence of both pipB and pipA. SARC strains 1, 2, 5, 6, 7, 9, 10, 15 and 16 give a strong hybridization signal, and SARC8 yielded two hybridization signals (Table 1). Only a weak signal was obtained for SARC13, and no signal was obtained with S. bongori (SARC11, SARC12) and S. enterica subspecies II strains (SARC3, SARC4) (Table 1). These results show that, in contrast to the left region of SPI-5 (Mirold et al., 2001), the right region is less conserved among the contemporary Salmonella lineages. This could indicate that the right region of SPI-5 was acquired after the divergence of S. enterica and S. bongori. Alternatively, SPI-5 might have been acquired by horizontal gene transfer before the divergence of Salmonella serovars. The right region may subsequently have been lost in the lineages with no selection pressure for a functional pipB and/or pipA, such as in S. bongori, which does not possess the SPI-2 TTSS required for PipB translocation.

Table 1. Distribution of pipB/pipA in Salmonella spp.
SARC no.Subspecies sigD/sopB a pipB/pipASPI-1 TTSSbSPI-2 TTSSc
  1. EcoRV-digested DNA from various isolates from the Salmonella reference collection C (SARC) was subjected to Southern blot analysis by hybridization with a pipB–pipA probe.

  2. + indicates a strongly hybridizing signal, ± denotes a weak hybridization signal, and – indicates that no signal was obtained.



  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

This work establishes for the first time that effectors encoded within a single PAI can be induced by distinct regulatory cues and translocated by different TTSS. It is important to note that, despite the expression of SigD/SopB and PipB being controlled by different regulatory cues, each is co-ordinately regulated with their dedicated TTSS (Fig. 6). Thus, SPI-5 demonstrates the convergence of co-ordinate expression of an effector with its dedicated TTSS. S. Typhimurium has evolved to not only export some proteins through both TTSS (Miao and Miller, 2000) but also to translocate effectors encoded on one PAI via different TTSS. This is also the first instance of a direct interaction between three chromosomal PAIs – in this instance, SPI-1, SPI-2 and SPI-5. The integrated regulation of horizontally acquired genes, regardless of their genomic location, is also likely to be evident for other pathogenic bacteria that encode two TTSS dedicated to the translocation of effectors, including Yersinia pseudotuberculosis (Cornelis and Van Gijsegem, 2000; Haller et al., 2000) and enterohaemorrhagic Escherichia coli (Perna et al., 2001). Our findings may have even wider implications, given that the flagellar biosynthetic apparatus has recently been shown to be capable of secreting virulence factors (Young et al., 1999).


Figure 6. Clustering of differentially regulated and translocated effectors within SPI-5. SPI-5 is located at 24 centisomes on the S. Typhimurium chromosome (McClelland et al., 2001). SPI-5-encoded genes are shaded in grey or black. SigD/SopB is expressed under SPI-1-inducing conditions and is translocated by the SPI-1 TTSS to effect membrane ruffling (Zhou et al., 2001) and host cell signalling (Steele-Mortimer et al., 2000). pipB is transcribed from a separate promoter under SPI-2-inducing conditions. PipB is subsequently translocated by the SPI-2 TTSS to the SCV and Sifs. The non-uniform base composition within SPI-5 suggests that this PAI may be a mosaic structure. The right region comprising pipB–pipA, which is not present in all Salmonella spp., has a lower (G + C) content compared with the left region. The DNA base composition at the boundaries of SPI-5 is distinctly different from both regions of SPI-5. As is characteristic of many PAIs, SPI-5 is bordered by a transfer RNA gene, in this case tRNASer.

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We have also shown that the genetic organization of SPI-5 is variable, as pipB/pipA are not found in all Salmonella spp., unlike sigD/sopB. SPI-1 (Zhou et al., 1999b), SPI-2 (Hensel et al., 1999), SPI-3 (Blanc-Potard et al., 1999) and SPI-4 (Wong et al., 1998) are proposed to be mosaic structures. The heterogeneous base composition of SPI-5 suggests that it is also a composite structure (Fig. 6). Although the S. Typhimurium LT2 and S. Typhi CT18 genomes have a G + C content of 53% and 52.1%, respectively (McClelland et al., 2001; Parkhill et al., 2001), the overall G + C content of SPI-5 from these serovars is 43.6% and 43.4% respectively (McClelland et al., 2001; Parkhill et al., 2001). The region comprising pipD–sigE (left region of SPI-5) has a G + C content of 46.2%, whereas the right region (pipB–pipA) has a significantly lower G+C content of 39.2% (Fig. 6). Such differences in base composition imply that these regions were acquired as separate genetic elements. However, it is important to note that the right region of SPI-5 has been conserved in Salmonella lineages (i.e. S. enterica subspecies I), in which pipB and pipA contribute to Salmonella virulence (Wood et al., 1998). Likewise, another SsrA/SsrB-dependent effector, SspH2, which is not encoded within SPI-2 but is translocated by the SPI-2 TTSS, is also absent from S. bongori serotypes (Tsolis et al., 1999). Together, these observations support the notion that the SPI-2 TTSS co-evolved with its translocated effectors, after the divergence of S. enterica from S. bongori. This is analogous to the evolutionarily earlier acquisition of the ‘invasion virulon’ (Mirold et al., 2001), which comprises the SPI-1 TTSS, and its secreted effectors required for host cell invasion, sopE, sopE2 and sopB/sigD. It has been proposed that such a multistep acquisition of different virulence genes contributed to Salmonella speciation and host adaptation (Baumler et al., 1998).

Like other SPI-1 and SPI-2 TTSS targeted effectors, PipB translocation signals localize to its N-terminal domain. However, the deduced amino acid sequence of PipB does not contain the recently identified N-terminal motif that is recognized by both the SPI-1 and SPI-2 TTSS (Beuzon et al., 2000; Brumell et al., 2000; Guy et al., 2000; Miao and Miller, 2000). As such, it remains to be determined how PipB is recognized and translocated by the SPI-2 TTSS. Searches of S. Typhimurium and S. Typhi genome sequencing projects ( and against the N-terminal 200 amino acids of PipB identified only one ORF in both databases, a previously unidentified gene that we have designated pipB2. PipB and PipB2 share 33% identity and 67% similarity distributed over their entire sequence. We are currently investigating whether PipB2 is also a Salmonella-translocated protein. It is becoming increasingly evident that the Salmonella genome encodes a number of effectors with structurally similar homologues, for example sopE/sopE2 (Hardt et al., 1998; Stender et al., 2000) and sspH1/sspH2 (Miao et al., 1999). Whether such duplication results from divergent or convergent evolution remains an interesting, but unanswered, question. In these instances, the two similar effectors serve redundant and/or additive functions. Although PipB contributes to secretory and inflammatory responses in bovine enteropathogenesis (Wood et al., 1998), it has no major effect on the development of systemic disease in mice (Wood et al., 1998) (Fig. 5B), nor does PipB contribute to host cell invasion (Pfeifer et al., 1999) or intracellular replication in phagocytic cells (Fig. 5A). Although we currently have no defined role for PipB in Salmonella pathogenesis, it is possible that, as for some other Salmonella effectors, PipB and PipB2 share overlapping functions. If this is the case, a ΔpipBΔpipB2 mutant is more likely to provide insight into the exact function of PipB.

A transiently transfected SifA–green fluorescent protein (GFP) chimera co-localizes with Sifs in epithelial cells (Beuzon et al., 2000) and is sufficient to form Sifs in these cells (Brumell et al., 2001). Although the localization of bacterially translocated SifA has yet to be demonstrated, SifA is translocated by the SPI-2 TTSS and is proposed to be targeted to the cytosolic face of the SCV membrane (Beuzon et al., 2000). Here, we show that PipB is a Salmonella-secreted protein that also localizes to the SCV and Sifs. However, unlike SifA, PipB does not appear to be required for Sif formation. However, at this point, we cannot confirm an indirect role for PipB in Sif or SCV homeostasis. Although the purpose of Sifs in Salmonella pathogenesis is unknown, one possible function is to provide a framework for the attachment of Salmonella effectors, including PipB and SifA. The association of SPI-2 translocated effectors with the Sif and SCV membrane would localize such bacterial proteins to interface directly with host cell functions and maintain the SCV. Determining the intracellular localization and actions of other SPI-2 translocated effectors will reveal whether Sifs do indeed play such a scaffolding role.

In order for bacterial pathogens to benefit from the acquisition of foreign DNA via lateral transfer, they must integrate the functioning of the new gene(s) with the rest of the genome. Here, we show that one SPI (SPI-5) has probably acquired DNA sequences from different sources to encode effectors that are differentially expressed and targeted by distinct TTSS. The co-ordinate expression of these effectors with their dedicated TTSS, at least in part, dictates the specificity of this interaction. The functional and regulatory cross-talk between three chromosomal PAIs, SPI-1, SPI-2 and SPI-5, highlights how PAIs have co-evolved to generate an integrated network of virulence functions. Such a concept impacts significantly on the evolution of PAIs and bacterial pathogenesis in general.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Bacterial strains

The bacterial strains and plasmids used in this study are detailed in Table 2. Strains from the SARB (Boyd et al., 1993) and SARC (Boyd et al., 1996) have been described previously. To construct a ΔpipB mutant, a non-polar in frame deletion cassette of pipB (corresponding to a deletion of amino acids 14–283) was ligated into the positive selection suicide vector pRE112 (Edwards et al., 1998) and transformed into E. coli SY327λpir. The ΔpipB deletion mutant was then constructed by allelic exchange into S. Typhimurium SL1344 or SL1344 ΔssaR and confirmed by PCR analysis. ΔpipB invA::kan and ΔpipB ssrB::kan strains were constructed by P22 transduction from SL1344 invA::kan (SB103) (Galan and Curtiss, 1991) and S. Typhimurium 14028s ssrB::kan, respectively (a kind gift from F. Heffron), to strain SL1344 ΔpipB. To construct a ΔpipA mutant, a deletion cassette of pipA (internal deletion of amino acids 11–103 followed by a premature stop codon) was ligated into pRE112, and selection was continued as described above for ΔpipB. For complementation of the ΔpipB mutant, the ORF of pipB plus 547 bp of sequence upstream of the ATG start codon was amplified from S. Typhimurium SL1344 genomic DNA by PCR with Pfx polymerase (Gibco BRL) and the oligonucleotides pipB-C2 (see Table 3 for all oligonucleotide sequences) and pipB-C3. Purified PCR product was cloned into pACYC184 (New England Biolabs) to give pACB-1. A plasmid encoding PipB tagged at the C-terminus with two haemagglutinin epitopes was constructed by inverse PCR using pACB-1 as a template with the oligonucleotides pipBCHA-1 and pipB-2HA-R and Elongase (Gibco BRL). The resulting PCR fragment was digested with XhoI and self-ligated to produce plasmid pACB C-2HA. A plasmid encoding SigD tagged at the C-terminus with a myc tag was PCR amplified with LK004 and LK005 using pACDE (Marcus et al., 2001) as a template. The PCR product was digested with XhoI and self-ligated to give pACDE C-myc.

Table 2. Plasmids and bacterial strains used in this study.
Plasmid or strainDescriptionReference
Plasmids and cloning vectors
pACYC184CmR, TetRNew England Biolabs
pCMV-mycAmpR, mammalian expression vectorClontech
pEGFP-N1KanR, mammalian expression vectorClontech
pGEM T-EasyAmpR, vector for cloning PCR productsPromega
pRE112CmR, suicide vector containing sacB1 Edwards et al. (1998)
ΔpipB pRE112CmR, in frame deletion of pipB (amino acids 14–283) in pRE112This study
ΔpipA pRE112CmR, deletion and truncation of pipA (amino acids 11–103 followed by stop codon) in pRE112This study
pACB-1CmR, pipB coding sequence plus 547 bp upstream of ATG start in SalI–HindIII site of pACYC184This study
pACDECmR, sigDE coding sequence plus 415 bp upstream of ATG start in SalI–HindIII site of pACYC184 Marcus et al. (2001)
pACB C-2HACmR, encodes HA-tagged PipB in pACYCThis study
pACDE C-mycCmR, encodes myc-tagged SigD in pACYCThis study
pCMV-pipB pipB coding sequence cloned into EcoRI–BglII sites of pCMV-mycThis study
pEGFP-sigD29-207 sigD fragment (codons 29–207) in HindIII–BamHI site of pEGFP-N1S. L. Marcus (unpublished)
PipB N90-CyaApACYC184 expressing N-terminal 90 amino acids of PipB fused to CyaAThis study
PipB N120-CyaApACYC184 expressing N-terminal 120 amino acids of PipB fused to CyaAThis study
PipB N150-CyaApACYC184 expressing N-terminal 150 amino acids of PipB fused to CyaAThis study
PipB N180-CyaApACYC184 expressing N-terminal 180 amino acids of PipB fused to CyaAThis study
PipB N210-CyaApACYC184 expressing N-terminal 210 amino acids of PipB fused to CyaAThis study
E. coli strains
DH5αCloning strainGibco BRL
SM10λpirKanR; for mobilizing ΔpipB pRE112 and ΔpipA pRE112 into SL1344 Miller and Mekalanos (1988)
SY327λpirFor propagation of pRE112 Miller and Mekalanos (1984)
Salmonella strains
SL1344Wild type Hoiseth and Stocker (1981)
ΔsigDSL1344, in frame deletion of sigD Steele-Mortimer et al. (2000)
ΔpipBSL1344, in frame deletion of pipB (missing amino acids 14–283)This study
ΔpipASL1344, deletion of pipA (missing amino acids 11–103 followed by a stop codon)This study
E12A2SL1344 pipB::luxABtet Pfeifer et al. (1999)
SB103SL1344 invA::kan Galan and Curtiss (1991)
ΔpipB invA::kan pipB deletion mutant defective in SPI-1 TTSSThis study
ΔssaRSL1344, in frame deletion of ssaR Brumell et al. (2001)
ΔpipBΔssaR pipB deletion mutant defective in SPI-2 TTSS 14028sThis study
MJW112 ssrB::kanM.J. Worley and F. Heffron (unpublished)
ΔpipB ssrB::kan pipB deletion mutant defective in SPI-2 gene regulationThis study
Table 3. Sequences of oligonucleotides used in this study.
OligonucleotideSequence (5′ to 3′)
  1. Restriction sites are underlined.


Molecular biology techniques

Salmonella Typhimurium SL1344 was grown under SPI-1- or SPI-2-inducing conditions as described previously (Miao and Miller, 2000). Total bacterial RNA was isolated as described previously (Celli and Trieu-Cuot, 1998). Northern analysis of 10 μg of total RNA was by standard techniques. A 533 bp sigD fragment, PCR amplified with the oligonucleotides SM033 and SM026, was 32P-radiolabelled using the PrimeIt II kit (Stratagene), replacing the random primers with SM033. A probe comprising the ORF of pipB was amplified with pCMVpipB-F and pCMVpipB-R and 32P-radiolabelled using the PrimeIt II kit with pCMVpipB-R. Hybridization was carried out overnight at 42°C in ULTRAHyb buffer (Ambion). For RT–PCR analysis, total bacterial RNA was DNase (Ambion) treated, and 1 μg was reverse transcribed with a gene-specific primer and Superscript II reverse transcriptase (Gibco BRL) according to the manufacturer’s instructions. The gene-specific primers were: for pipD, pipD-R1; for sigD/sopB, sigD10; for pipB, pipBF6; and for pipA, pipA-rev. An aliquot (1/20th) of the cDNA reaction was subjected to 35 cycles of PCR amplification using AmpliTaq gold DNA polymerase (Perkin-Elmer) with the following oligonucleotides: for pipD, pipD-R2 and pipD-F1 (449 bp product); for sigD/sopB, sigD10 and sigD8 (675 bp); for pipB, pipBF6 and E– (789 bp); for pipA, pipA-rev and pipA-for (319 bp). For pipB transcript analysis, the 5′ RACE system (Gibco BRL) was used. The gene-specific primer for cDNA synthesis was E3615. dC-tailed cDNA was amplified by PCR for 40 cycles with the pipB-specific oligonucleotide E+ and the abridged anchor primer supplied with the kit. PCR products were cloned into the pGEM T-Easy vector (Promega) and sequenced to determine the transcript start site. Southern analysis was performed at 55°C as described previously (Mirold et al., 2001) using the Amersham ECL random prime labelling and detection kit. A probe comprising the ORF of pipB was amplified from S. Dublin chromosomal DNA with the oligonucleotides Southern-A and Southern-B. A pipB–pipA probe was amplified with the oligonucleotides Southern-A and Southern-C.

Analysis of PipB expression

For the analysis of PipB expression, bacteria were grown under SPI-1- and SPI-2-inducing conditions as described by Miao and Miller (2000). Protein from an equal number of bacterial cells, adjusted according to OD600 readings, was analysed by Western blot analysis. To examine PipB expression after bacterial invasion of cultured cell lines, HeLa (human cervical adenocarcinoma cell line, ATCC CCL2) or RAW 264.7 (murine macrophage-like cell line, ATCC TIB-71) cells were seeded in six-well plates and incubated overnight at 37°C in DMEM containing 10% heat-inactivated fetal calf serum (FCS) in 5% CO2. Bacteria were prepared, cells infected and samples collected as described previously (Steele-Mortimer et al., 2000). Concurrent invasion assays were carried out (Brumell et al., 2001) to enumerate intracellular bacteria over the infection time course. For Western analysis, samples were normalized to equivalent bacterial cfu for each time point. Western blots were performed as described previously (Steele-Mortimer et al., 2000). Blots were incubated overnight at 4°C in Tris-buffered saline containing 0.1% (v/v) Tween 20 (TBST)/5% (w/v) skimmed milk powder with monoclonal α-HA (Covance, 1:1000), monoclonal α-myc (Santa Cruz, 1:1000), monoclonal α-DnaK (Stressgen, 1:1000) or polyclonal α-SseB (1:1000) (a kind gift from Dr J. Brumell). Secondary anti-bodies, goat α-mouse horseradish peroxidase (HRP; Sigma, 1:5000) or goat α-rabbit HRP (Sigma, 1:5000), were applied in TBST/milk at room temperature for 1 h followed by detection with ECL reagent (Amersham Pharmacia).

Virulence tests and macrophage survival assays

Female BALB/c mice (6–8 weeks old) were obtained from Jackson Laboratories. Mice were kept in sterilized, filter-topped cages, handled in tissue culture hoods and fed autoclaved food and water under specific pathogen-free (SPF) conditions at our animal facilities. Sentinel animals were routinely tested for common pathogens. The protocols used were in direct accordance with guidelines drafted by the University of British Columbia’s Animal Care Committee and the Canadian Council on the Use of Laboratory Animals. Bacterial subcultures were grown to mid-log phase as described previously (Steele-Mortimer et al., 2000) and diluted in Luria–Bertani (LB) broth before inoculation. Groups of five mice were orally inoculated with ≈ 1 × 104 cfu in 0.1 ml of LB broth. To assess the virulence of the tested strains, mice were monitored throughout the course of infection, and any that showed extreme stress or became moribund were euthanized.

For macrophage survival assays, RAW 264.7 cells were grown as described above. Bacterial strains were grown shaking overnight in LB broth at 37°C. Stationary phase bacteria were diluted in DMEM–10% FCS to an OD600 of 1.0 and opsonized at 37°C for 30 min in 15% normal human serum. Bacteria were centrifuged onto macrophages seeded in 24-well tissue culture plates at a multiplicity of infection (MOI) of 5:1 and incubated for 25 min. Infected monolayers were washed three times with PBS to remove extracellular bacteria and incubated for 90 min in DMEM–10%FCS containing 50 μg ml–1 gentamicin. Thereafter, the gentamicin concentration was reduced to 10 μg ml–1 in DMEM–10% FCS. At either 2 h or 24 h p.i., cells were lysed in 1% (v/v) Triton X-100–0.1% (w/v) SDS and plated on LB agar to enumerate cfu.

PipB–CyaA fusions and cAMP assays

CyaA fusions to various N-terminal truncations of PipB were created. CyaA was amplified from pMS107 (Sory and Cornelis, 1994) with the oligonucleotides cyaA-Bgl and cyaA-Hind. The resulting fragment was digested with BglII and HindIII. N-terminal fragments of PipB were amplified from S. Typhimurium SL1344 genomic DNA with the forward primer pipB-Sal and one of the following reverse primers: pipB90-Bgl, pipB120-Bgl, pipB150-Bgl, pipB180-Bgl or pipB210-Bgl. These PCR fragments were subsequently digested with BglII and SalI. The CyaA and PipB fragments were ligated into pACYC184 digested with SalI and HindIII creating PipB–CyaA fusions expressed from the native pipB promoter. HeLa cells were infected with S. Typhimurium expressing the PipB–CyaA fusions as described previously (Steele-Mortimer et al., 2000). At 5.5 h p.i., HeLa cells were washed twice in PBS and lysed in lysis buffer 1B (supplied with the cAMP immunoassay kit) supplemented with 0.1 M HCl for 10 min at room temperature. Samples were subsequently neutralized with 1 M NaOH and stored at –20°C. cAMP in cell extracts was measured using a cAMP enzyme immunoassay system (Amersham Pharmacia Biotech) according to the manufacturer’s instructions. Protein content was determined using the Bio-Rad protein assay reagent.

Immunofluorescence and confocal microscopy

HeLa cells were grown on glass coverslips overnight. Infection with ΔpipB (pACB C-2HA) was carried out as described elsewhere (Steele-Mortimer et al., 2000). Cells were fixed at 8 h or 21 h p.i. and processed as described elsewhere (Steele-Mortimer et al., 1999). Primary antibodies were rabbit α-Salmonella LPS (Difco, 1:500), mouse α-HA (Covance, 1:1000) or rabbit α-lysosomal-associated membrane protein-2 (LAMP-2; Carlsson et al., 1988). Secondary antibodies were Alexa Fluor 568 goat α-rabbit IgG (Molecular Probes, 1:500) and Alexa Fluor 488 goat α-mouse IgG (Molecular Probes, 1:500). Samples were viewed using a Zeiss Axiovert S100 TV microscope attached to a Bio-Rad Radiance Plus confocal microscope with the 63× oil objective. Images of 512 × 512 pixels (102 × 102 μm) were acquired using Bio-Rad LASERSHARP software. Sections of 0.2 μm thickness were assembled into flat projections using NIH IMAGE and imported into ADOBEPHOTOSHOP.


  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Genome sequence data for S. Typhimurium LT2 and S. Typhi were obtained from the Washington University School of Medicine ( and The Sanger Centre ( We thank members of the Finlay laboratory and Rachel Fernandez for critical reading of this manuscript, and José Luis Puente for his endless advice and encouragement. We also thank Minoru Fukuda for providing the α-LAMP-2 antibody, John Brumell for the α-SseB antibody, and Fred Heffron for the ssrB::kan strain. B.A.V. is supported by a Canadian Digestive Disease Institute/Medical Research Council of Canada (MRC) postdoctoral fellowship and is an Honorary Izaak Walton Killam Fellow. Work in the laboratory of W.-D.H. is supported by a grant from the Deutsche Forschungsgemeinschaft. B.B.F. is an International Research Scholar of the Howard Hughes Medical Institute and a Distinguished Investigator of the Canadian Institute for Health Research. B.B.F. is supported by the Canadian Institute of Health Research and the Howard Hughes Medical Institute.


  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
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