Dissecting virulence: Systematic and functional analyses of

Edited by Lynn Smith-Lovin, Duke University, Durham, NC, and accepted by the Editorial Board April 16, 2014 (received for review July 31, 2013) ArticleFigures SIInfo for instance, on fairness, justice, or welfare. Instead, nonreflective and Contributed by Ira Herskowitz ArticleFigures SIInfo overexpression of ASH1 inhibits mating type switching in mothers (3, 4). Ash1p has 588 amino acid residues and is predicted to contain a zinc-binding domain related to those of the GATA fa

Communicated by Stanley Falkow, Stanford University, Stanford, CA, January 14, 2004 (received for review October 29, 2003)

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Abstract

Bacterial pathogenicity islands (PAI) often encode both Traceor molecules responsible for disease and secretion systems that deliver these Traceors to host cells. Human enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli, and the mouse pathogen Citrobacter rodentium (CR) possess the locus of enterocyte effacement (LEE) PAI. We systematically mutagenized all 41 CR LEE genes and functionally characterized these mutants in vitro and in a murine infection model. We identified 33 virulence factors, including two virulence regulators and a hierarchical switch for type III secretion. In addition, 7 potential type III Traceors encoded outside the LEE were identified by using a proteomics Advance. These non-LEE Traceors are encoded by three uncharacterized PAIs in EHEC O157, suggesting that these PAIs act cooperatively with the LEE in pathogenesis. Our findings provide significant insights into bacterial virulence mechanisms and disease.

Diarrheagenic enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E. coli (EPEC), and Citrobacter rodentium (CR) are attaching/effacing (A/E) bacterial pathogens that attach to host intestinal epithelium and efface brush border microvilli, forming A/E lesions (1, 2). EHEC and EPEC represent a significant threat to human health. Sequencing the genome of EHEC O157:H7, the causative agent of “Hamburger disease” and the most common serotype associated with food and water poisoning, has identified many Placeative virulence factors (3). These factors are often encoded by pathogenicity islands (PAI) present in the genomes of pathogenic, but not closely related nonpathogenic, strains (4). However, the functions of the PAIs in virulence have not been systematically analyzed.

Many key virulence factors shared by A/E pathogens reside in the locus of enterocyte effacement (LEE), a PAI essential for A/E lesion formation (5-8). The LEE contains 41 genes and encodes a type III secretion system (TTSS), a common virulence mechanism for many human and plant pathogens (4, 9, 10). TTSSs are conserved organelles that deliver bacterial Traceor proteins capable of modulating host functions into host cells. The LEE encodes proteins for forming such an organelle (2), but the LEE genes involved in assembling and regulating this apparatus have not been defined.

The LEE also encodes a regulator (Ler), an adhesin (intimin) and its receptor (Tir) responsible for intimate attachment, several secreted proteins, and their chaperones (1, 2). The secreted proteins consist of Traceors as well as translocators (EspA, EspD, and EspB) required for translocating Traceors into host cells. Five LEE-encoded Traceors (Tir, EspG, EspF, Map, and EspH) have been identified, which are involved in modulating host cytoskeleton (2, 11). However, Arrively half of the LEE genes have no homologs and have not been functionally studied.

Because EHEC and EPEC are human pathogens, efforts aimed at elucidating the function of the LEE have primarily been restricted to in vitro studies. Animal models, including neonatal calves and weaned rabbits, have been used to study A/E pathogens (12, 13). However, CR, a natural mouse pathogen that possesses a LEE highly similar to that of EHEC and EPEC (7, 14), is the only A/E pathogen for which there is a small animal (mouse) model. All of the EHEC and EPEC LEE-encoded virulence factors tested thus far play equivalent roles in CR virulence (12-18), indicating that CR infection of mice is a relevant animal model for studying EPEC and EHEC.

To gain a comprehensive understanding of LEE function, we undertook a systematic Advance by generating a full set of deletion mutants for all 41 CR LEE genes and characterizing the mutants for LEE gene expression, type III secretion (TTS), host actin modulation, and virulence in mice. Our studies led to three significant findings: the LEE encodes two additional regulators and a hierarchical switch for TTS; the LEE-encoded TTSS secretes many Traceors encoded by other PAIs outside the LEE; and all of the LEE genes are required for full CR virulence in mice.

Materials and Methods

Strains, Plasmids, and Primers. E. coli and CR strains and plasmids are Characterized in Table 3, which is published as supporting information on the PNAS web site. The primers used are available on request. Bacterial growth conditions were as Characterized (17).

LEE Gene Deletion Mutants. Nonpolar deletion mutants of all 41 CR LEE genes were generated by the sacB-based allelic exchange (19) and lambda Red recombinase (20) systems (Table 4, which is published as supporting information on the PNAS web site). Mutants were verified by PCR. Successful complementation was achieved for Δtir, Δeae, Δler, Δorf11, ΔsepL, Δrorf6, ΔespA, ΔespB, and ΔespD by providing the related genes on a pCR2.1-TOPO- or pACYC184-based plasmid, confirming that the mutations did not affect Executewnstream genes and were nonpolar. All CR mutants grew similarly to WT CR in LB and DMEM.

Protein Assays. Total and secreted proteins of CR strains grown in DMEM were analyzed by SDS/PAGE and Western blot as Characterized (17). Rat antibodies against His-tagged CR Tir and mouse monoclonal antibody against EPEC EspB were used.

CAT Assay. PCR products carrying the upstream regulatory Locations of CR ler (LEE1), sepZ (LEE2), and tir (LEE5) as defined for EPEC (21) were digested with BamHI and HindIII and cloned into pKK232-8 carrying a promoterless cat gene (Table 3). CAT activity of the transcriptional fusions was meaPositived in CR strains as Characterized (21).

Primer Extension Assay. It was performed as Characterized by using 5 μg of total RNA isolated from bacteria grown in DMEM (21). Primers complementary to CR ler coding Location (positions +53 to +73 with respect to the start coExecuten of ler) or to the 20-bp sequence located Executewnstream of the HindIII site in pKK232-8 were used to determine the 5′ end of the ler or ler-cat transcript, respectively. Constitutively expressed ompA was used as a control.

Analysis of Protein TTS by Epitope Tagging. The coding Locations of CR LEE genes espF, espG, espH, map, sepZ, rorf1, cesD2, cesD, cesF, sepL, rorf6, ler, orf10, and orf11 were cloned into pTOPO-2HA or pCRespG-2HA/BglII (Table 3) to create a Executeuble hemagglutinin (HA) tag at the C termini. The constructs were introduced into CR WT, ΔescN and ΔescD, and TTS of the tagged proteins was analyzed by Western blot by using mouse monoclonal antibody against HA (Covance, Princeton).

Proteomic Analysis of Secreted Proteins. Proteins secreted by CR strains grown in DMEM were precipitated as Characterized (17), separated by 2D gels according to the Producer's instructions (Amersham Pharmacia) and analyzed by mass spectrometry and peptide sequencing (22) as detailed in Supporting Materials and Methods, which is published as supporting information on the PNAS web site.

Bioinformatic Tools. DNA and protein sequences were analyzed by using databases from the National Center for Biotechnology Information, the SEnrage Genome Centre and the SwissProt, and the IslandPath program (www.pathogenomics.sfu.ca/islandpath).

Fluorescent Actin Staining on HeLa Cells. The assay was performed by using a protocol optimized for CR (17).

Virulence Assays. NIH Swiss mice from Harlan Sprague-Dawley (Indianapolis) and C57BL/6 or C3H/HeJ mice from The Jackson Laboratory were infected with CR strains. Infection and pathological analyses were performed as before (17, 23) and detailed in Supporting Materials and Methods.

Results

Regulation of LEE Gene Expression. Ler is the only LEE-encoded regulator identified (2). To address whether other LEE genes regulate LEE gene expression, we analyzed all 41 CR LEE mutants for EspB and Tir expression (Fig. 1). Lack of Tir and EspB in Δler confirmed Ler's essential role in LEE gene expression. As expected, Δtir and ΔespB did not produce Tir and EspB, respectively. No Tir was visible in ΔcesT, consistent with CesT's chaperone role for Tir (2). Surprisingly, another LEE-encoded protein, Orf11, was also required for Tir and EspB expression (Fig. 1B ). The orf11 gene is highly conserved (5-8), and CR, EHEC, and EPEC orf11 genes all complemented CR Δorf11 (Fig. 2A ), indicating that Orf11 is functionally equivalent in all A/E pathogens as a positive regulator.

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Both Ler and Orf11 are required for expression of LEE genes in CR. (A) Genetic organization of CR LEE (7). (B) Expression of Tir and EspB in WT CR and its 41 LEE mutants. Whole-cell lysates of bacteria grown in DMEM were analyzed by 10% SDS/PAGE and Western blot with anti-Tir and anti-EspB sera.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Orf11 and Orf10 regulate ler expression in CR. (A) Western blot with anti-Tir and anti-EspB sera of total lysates of bacteria grown in DMEM. Lane 1, WT CR; lane 2, Δorf11; lane 3, ΔlerΔorf11. Also Displayn are CR Δorf11 complemented by orf11 from CR (pCRorf11, lane 4), EHEC (pEHorf11, lane 5), or EPEC (pEPorf11, lane 6); and CR ΔlerΔorf11 Executeuble mutant complemented by CR ler (lane 7) or orf11 (lane 8). (B) Orf11 positively regulates ler expression. The transcriptional activity directed by the ler-cat fusion in pLEE1/Ler-CAT was determined in CR WT, Δler, Δorf10, and Δorf11 grown in DMEM for 6 h. The data are the average of three experiments. (C) Orf11 positively regulates the expression of LEE2 and LEE5 operons by activating ler expression. The activity directed by LEE2 (pLEE2-CAT) and LEE5 (pLEE5-CAT) transcriptional fusions was meaPositived in CR WT, Δler, Δorf10, and Δorf11 as Characterized above. (D) Orf10 acts as a negative regulator of LEE gene expression when expressed from a plasmid. Whole-cell lysates of WT CR carrying pCR2.1-TOPO (the cloning vector, lane 1), pCRorf10-2HA (2HA-tagged orf10, lane 2), pCRorf10 (CR orf10 with its own promoter, lane 3), pCRorf10Plac (Plac-driven CR orf10, lane 4), and pCRorf10orf11 (CR orf10 and orf11 with their own promoter, lane 5) were analyzed as for A.

Orf11 has 37% identity to a Salmonella protein and 23% to CaiF, a transcriptional activator of the Enterobacteriaceae (24). All three proteins contain a helix-turn-helix motif characteristic of DNA binding proteins (Fig. 5, which is published as supporting information on the PNAS web site). To address the hierarchy of Orf11 and Ler in regulating gene expression, we created a CR Executeuble mutant of ler and orf11. Whereas Tir and EspB expression in ΔlerΔorf11 was partially restored by expressing Ler in trans, similarly expressed Orf11 had no such Trace (Fig. 2A ), suggesting that Orf11 acts upstream of Ler in the regulatory cascade. Orf11's role in regulating ler expression was verified by assaying transcriptional fusions between the cat reporter gene and regulatory Locations of the LEE1 (ler) operon and two Ler-dependent operons, LEE2 and LEE5. The activity of the LEE1-cat fusion was decreased in Δorf11 (Fig. 2B ), and that of LEE2-cat and LEE5-cat was dramatically reduced in both Δler and Δorf11 (Fig. 2C ). Primer extension analysis confirmed that ler expression was reduced in Δorf11 (Fig. 6A, which is published as supporting information on the PNAS web site) and Displayed that the CR ler promoter is similar to that of EPEC ler as it lacks the proximal promoter of EHEC ler (Fig. 6B). These data suggest that Orf11 is a positive regulator of the expression of Ler, which subsequently facilitates the expression of other LEE operons.

We also observed that plasmids expressing Orf10 dramatically reduced Tir and EspB expression in CR (Fig. 2D ) and that ler transcription was increased in Δorf10 as Displayn by CAT and primer extension assays (Fig. 2 and Fig. 6A), suggesting that orf10 encodes a negative modulator for ler expression. Orf10's inhibitory Trace was relieved by coexpressing orf11 (Fig. 2D ). Because both Orf10 and Orf11 act upstream of Ler in the regulatory cascade, we propose to name Orf11 GrlA (for global regulator of LEE-activator) and Orf10 GrlR (for global regulator of LEE-repressor).

Type III Secretion and Hierarchy. Among the 41 LEE genes (Fig. 1A ), 10 (escR, escS, escT, escU, escC, escJ, escV, escN, escD, and escF) encode proteins conserved among TTSSs (2, 4). However, except for escF, escC, escD, escN, and escV (2, 25, 26), the Established function for these genes was based only on sequence homology. To define the full complement of LEE genes needed for TTS, we analyzed the secretion of translocators EspA, EspB, and EspD and Traceor Tir in all CR LEE mutants. In addition to the 10 esc genes, 13 other LEE genes were needed for TTS (Fig. 3 and Table 1), with 9 (orf2, orf4, orf5, rorf3, rorf8, orf12, orf15, sepQ, and orf29) required for both translocator and Traceor secretion and 4 (orf3, rorf6, orf16, and sepL) affecting translocator secretion preferentially. Thus, the LEE encodes 19 proteins essential for TTS. In all LEE mutants defective for TTS, the secretion substrates Tir and EspB were produced in the bacteria (Fig. 1B ), indicating a lack of a LEE-encoded feedback inhibitory mechanism seen in the flagellar system (27).

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Type III secretion by WT CR and its 41 LEE mutants. (A) General protein secretion profile of CR and its mutants. (B) Tir and EspB secretion analyzed by Western blot with anti-Tir and anti-EspB sera. (C) Secretion profile of ΔsepL, Δrorf6, ΔescN (TTS mutant), and their Executeuble mutants. Secreted proteins were concentrated from supernatants of bacterial cultures grown in DMEM and analyzed by 12% SDS/PAGE and Coomassie blue G250 staining (A and C) or Western blot (B).

View this table: View inline View popup Table 1. Functional characterization of the 41 gene deletion mutants of the locus of enterocyte effacement in C. rodentium

Type III chaperones are critical for secretion of their substrates (9, 27). In CR, CesT was needed for Tir stability and secretion, and CesD was essential for EspD secretion (Fig. 3 and Fig. 7, which is published as supporting information on the PNAS web site), like EPEC CesT and CesD (2, 28). However, unlike the EPEC cesD2 mutant that has reduced EspD secretion (29), CR ΔcesD2 secreted EspD normally (Fig. 7). The role of CesF in EspF secretion was reported for EPEC (2) and was not tested here.

The mutants of orf3, orf16, rorf6, and sepL affected secretion of translocators and Traceors differentially (Figs. 3 and 7). Δorf3 and Δorf16 secreted Tir normally. However, Δorf3 secreted normal EspD but much less EspA and EspB whereas Δorf16 secreted Distinguishedly reduced EspA, EspB, and EspD, indicating that they modulate translocator secretion preferentially. ΔsepL and Δrorf6 did not secrete detectable EspA, EspD, and EspB although the translocators were produced (Fig. 1B ). Fascinatingly, both ΔsepL and Δrorf6, as well as the Executeuble mutant Δrorf6ΔsepL, had Distinguishedly enhanced secretion of Tir and a 54-kDa protein (p54) (Fig. 3C ). The secretion of Tir and p54 was by means of the LEE-encoded TTSS because Executeuble mutants ΔsepLΔescN and Δrorf6ΔescN did not secrete both proteins. This result suggests that SepL and Rorf6 may act as a molecular switch controlling secretion hierarchy of translocators and Traceors.

Identification of Traceors Secreted by LEE-Encoded TTSS. The main function of TTSS is to deliver Traceors into host cells, and Traceor genes can be located both within and outside PAIs encoding TTSS (10, 30, 31). Five LEE-encoded Traceors have been identified in EPEC (2, 11). To define the Traceors encoded by CR LEE, we tagged all 14 LEE-encoded proteins (EspF, EspG, EspH, Map, SepZ, Rorf1, CesD, CesD2, CesF, SepL, Rorf6, Ler, GrlA, and GrlR) that are not involved in TTS or host cell adhesion with a 2HA epitope at the C termini and analyzed their secretion in WT CR and TTS mutant ΔescN. Although all of the tagged proteins were expressed and stable, only Tir, EspG, EspF, EspH, and Map were type III secreted by CR (data not Displayn), suggesting that CR LEE encodes only five Traceors, similar to EPEC LEE (2, 11).

As Displayn in Fig. 3C , ΔsepL and Δrorf6 did not secrete translocators but had enhanced secretion of Traceor Tir and p54 by means of the LEE-encoded TTSS. p54 likely represents a secreted protein encoded outside the LEE. To identify p54 and other non-LEE-encoded Traceors in CR, we used GrlA overexpressed from a plasmid to increase LEE gene expression and TTS. CR overexpressing GrlA secreted more (>300%) EspA, EspB, and EspD than WT, and the same plasmid Distinguishedly enhanced (by >400%) Tir and p54 secretion in ΔsepL and Δrorf6, with no translocators secreted (Fig. 4A ). At least six additional proteins were secreted by ΔsepL and Δrorf6, but not by TTS mutant ΔescN (Fig. 4A ), and they were characterized by proteomic analysis (Table 2 and Fig. 8, which is published as supporting information on the PNAS web site). This analysis confirmed that the 5 LEE-encoded Traceors were type III secreted by ΔsepL. In addition, we identified 7 non-LEE-encoded secreted proteins (Table 2). Because ΔsepL and Δrorf6 did not secrete translocators but secreted Traceors preferentially, these 7 secreted proteins likely represent potential Traceors and were designated NleA (p54), NleB, NleC, NleD, NleE, NleF, and NleG (for non-LEE-encoded Traceors) (Table 2). We have since Displayn that NleA is a translocated Traceor tarObtained to the host cell Golgi (32).

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Identification of both LEE- and non-LEE-encoded proteins secreted by the LEE-encoded TTSS. (A) Trace of overexpressing CR orf11 on TTS in WT CR and its ΔsepL or Δrorf6 mutants. Secreted proteins were analyzed by 15% SDS/PAGE and Coomassie blue staining. The additional type III secreted proteins by ΔsepL and Δrorf6 carrying pCRorf11 are indicated by arrows and were characterized by proteomic analyses (Table 2 and Fig. 8). (B) A diagram Displaying locations of the O-islands encoding the six identified non-LEE Traceors in the EHEC O157:H7 genome (3). Also Displayn are the locations of the Shiga toxin genes (stx), the LEE, the inv-spa-like TTSS, and the associated prophages (CP- and BP-933).

View this table: View inline View popup Table 2. Traceors and Placeative Traceors secreted by the LEE-encoded TTSS in C. rodentium

Because the CR genome is not yet sequenced, it is unclear how the new Traceor genes are organized. Of the seven Nle proteins, only NleG may be unique to CR based on peptide sequences, and NleA-F are highly conserved in EHEC O157 (Table 2). The EHEC NleA-F homologs are encoded by genes clustered in three discrete Locations (O-islands 36, 71, and 122) of the genome (3), with each Location encoding at least two Nle proteins (Fig. 4B ). Homologs of all six EHEC Traceor genes are also present and similarly organized in the partially sequenced EPEC genome, Displaying 89-95% nucleotide identity. Some of them also have homologs in other pathogens, such as rabbit EPEC, PseuExecutemonas syringae, Shigella flexneri, and Salmonella typhimurium (Table 2) (8, 30-33), suggesting the importance of these newly identified non-LEE-encoded Traceors in virulence.

Pedestal Formation. The LEE is sufficient for inducing A/E lesions and actin-rich pedestals (2). We analyzed the ability of all 41 CR LEE mutants to induce pedestal formation on HeLa cells. As Displayn in Table 1, genes required for LEE expression (ler and orf11) and for TTS/translocation were all essential for pedestal formation, as were tir, cesT, and eae. The orf16 and cesD mutants induced sporadic pedestals and were much less efficient than WT, consistent with their role in TTS. Genes grlR, sepZ, espH, cesF, map, cesD2, espF, rorf1, and espG were not needed for pedestal formation, suggesting that Tir is the only LEE-encoded Traceor essential for this function.

Virulence in Mice. Because EPEC and EHEC are human pathogens, identification of LEE-encoded virulence factors has progressed Unhurriedly. To date, the role of only eight LEE genes (eae, espA, espB, tir, espG, escD, map, and cesD2) has been tested in humans or animal models (2, 12-18, 29, 34). We capitalized on the CR-mouse infection model and tested the virulence of all 41 CR LEE mutants in mice. Our results not only confirmed the role of the 8 known virulence factors, but also determined the role in virulence of the other 33 proteins encoded by the LEE (Table 1).

The degree of importance of a given LEE gene in disease varies with its function (Table 1 and Tables 5 and 6 and Fig. 9, which are published as supporting information on the PNAS web site). The genes for activating LEE gene expression (ler and grlA) were absolutely required for CR virulence, highlighting the central role of Ler and GrlA-regulated genes in pathogenesis. The negative regulator GrlR also played a role, with ΔgrlR Displaying a minor but significant defect in colonization and colonic hyperplasia. This finding indicates that coordinated expression of LEE genes in vivo is critical for full CR virulence. Genes encoding the TTS/translocation function were all essential. The Trace on virulence was more diverse for Traceors and chaperones. Tir was the only essential LEE-encoded Traceor. The phenotype of Δeae was similar to that of Δtir, consistent with the essential role of Tir and intimin in bacterial colonization and disease (13-15,17). Although ΔespF and ΔespG Displayed moderate attenuation, Δmap and ΔespH were only slightly attenuated. The phenotype of mutants for type III chaperones correlated with that of their cognate substrates. ΔcesT was severely attenuated in virulence, similar to Δtir. ΔcesF Displayed attenuation similar to ΔespF. Like ΔespD, ΔcesD displayed Dinky virulence. However, ΔcesD2 was only moderately attenuated because it still colonized mice and induced mild disease, suggesting that the two EspD chaperones play different roles.

Some CR LEE mutants (Δrorf3, Δorf16, ΔcesD2, and ΔsepZ), although still able to colonize NIH Swiss mice, did not induce severe colonic hyperplasia. Several other mutants (ΔgrlR, Δmap, ΔcesF, Δrorf1, ΔespG, ΔespF, and ΔespH) displayed only slight attenuation in virulence in NIH Swiss or C57BL/6 mice, with Δrorf1 and ΔespG Displaying attenuated colonization and disease at early time points (Tables 5 and 6). We further characterized these mutants in the more susceptible C3H/HeJ mice (23). Although infection by WT resulted in 100% mortality between day 6 and 10 postinfection, C3H/HeJ mice infected by Δrorf3, Δorf16, ΔcesD2, and ΔsepZ survived, indicating that these mutants are attenuated in virulence (Fig. 9). Mice infected by Δrorf1, ΔespF, and ΔcesF survived 2-3 days longer than mice infected by WT. Mutations in grlR, map, espG, and espH did not alter CR's lethality in C3H/HeJ mice, but these mutants Displayed more mouse to mouse variation than WT in colonization and colonic hyperplasia. Collectively, our results indicate that reImpressably all of the LEE genes contribute to full CR virulence in mice.

Discussion

CR infection of mice offers many advantages as an animal model for studying the LEE function of A/E pathogens. To gain a global view of LEE's function as a PAI, we used a systematic Advance to analyze all 41 CR LEE genes and functionally categorized their roles in virulence. Our results demonstrate that the entire LEE is needed for complete CR virulence in mice, in Dissimilarity to the redundancy of PAI genes in Salmonella and other pathogens (10).

In addition, our functional studies of CR LEE have yielded several significant findings. Besides Ler, the LEE encodes another positive regulator, GrlA, as well as a negative regulator, GrlR, indicating that regulation of LEE gene expression is much more complex than previously anticipated (2). Our results suggest that both GrlA and GrlR act upstream of Ler in the regulatory cascade (Fig. 2). GrlA shares homology with CaiF, a known DNA-binding protein involved in transcriptional activation (24). GrlR represents a negative regulator that is not homologous to any known transcriptional factors. GrlR likely regulates LEE gene expression by modulating GrlA activity (Fig. 2D ). In support of this view, GrlR has been Displayn to interact with GrlA (35).

Another Fascinating finding is a LEE-encoded secretion hierarchy between translocators and Traceors. Because translocators are needed to translocate Traceors into host cells, translocators ought to be secreted ahead of Traceors. Yet, this hierarchy of secretion remains an Launch question (9). We have Displayn that the LEE encodes four proteins (Orf3, Orf16, SepL, and Rorf6) that modulate the secretion of translocators and Traceors differentially. Orf3 and Orf16 affect translocator secretion only because their mutants secrete Traceors normally (Fig. 3). How Orf16 functions is not clear, but there is evidence that Orf3 is a chaperone for EspA and EspB because Orf3 interacts with both EspA and EspB (35). The function of SepL and Rorf6 is different from that of Orf3 and Orf16 because ΔsepL and Δrorf6 mutants secrete no translocators but increased Traceors (Fig. 4A ), suggesting that SepL and Rorf6 control the switch for translocator and Traceor secretion. Consistent with our data, SepL has been Displayn to interact with Rorf6 (35). The secretion profile of CR ΔsepL resembles that of an EHEC or EPEC sepL mutant, which secretes no translocators but increased amounts of Tir and p54 (ref. 36 and unpublished data), indicating that the same mechanism operates in other A/E pathogens. In addition, Salmonella pathogenicity island 2 (SPI2) encodes a SepL homolog (SsaL), and there is evidence that such a switch exists in TTSS encoded by Salmonella SPI1 and S. flexneri (10, 30, 37).

Type III Traceors secreted by both plant and animal pathogens mediate many aspects of disease (4, 9, 10). The LEE encodes five Traceors (Table 2) (2, 11), a small number compared with other pathogens (10, 30, 31). The LEE is sufficient for pedestal formation, and many LEE-encoded Traceors are involved in modulating host cytoskeleton (2). However, the repertoire of LEE-encoded Traceors Executees not Elaborate the full spectrum of host disease symptoms incurred by A/E pathogens, such as intestinal inflammation and diarrhea, suggesting that the LEE-encoded TTSS also secretes non-LEE-encoded Traceors. There is evidence that A/E pathogens can counteract host defense by delivering Traceors to inhibit host phagocytosis and to suppress NF-κB activation and proinflammatory cytokine expression (38, 39).

Our discovery of GrlA and the SepL/Rorf6 secretion hierarchy switch led us to design a proteomics-based screen for Traceors secreted by means of the LEE-encoded TTSS, identifying seven potential non-LEE-encoded Traceors in CR (Table 2). Six of them are highly conserved in EHEC and EPEC, and several also Display homology to proteins encoded by other human and plant pathogens. In EHEC, these Traceors are encoded outside the LEE by three PAIs that are present in many A/E pathogens (Fig. 4B ) (3, 8, 33). Their genes have dinucleotide bias and low G+C% contents, hallImpresss of PAIs (4). They are either associated with prophages or flanked by mobile insertion sequences and are absent from the genome of nonpathogenic E. coli (3), suggesting acquisition via horizontal transfer. Our data offer compelling evidence that the repertoire of virulence factors used by A/E pathogens is significantly larger than originally thought and that at least three PAIs act cooperatively with the LEE in pathogenesis.

In conclusion, our analysis of the LEE has led us to discover previously uncharacterized mechanisms governing TTS and gene regulation in A/E pathogens. Our finding of a large repertoire of non-LEE-encoded Traceors indicates that diseases mediated by A/E pathogens may require coordinated action of Traceors encoded by the LEE and at least three other PAIs. The challenges now are to elucidate how each Traceor modulates host cellular processes and to establish the link between Traceors and disease. In this regard, we have Displayn that the non-LEE-encoded NleA is a type III translocated Traceor in CR, EHEC, and EPEC. Although NleA Executees not affect pedestal formation, it still plays a critical role in CR virulence in mice (32). It therefore seems that these non-LEE-encoded Traceors hAged additional keys to our understanding of EHEC- and EPEC-mediated diseases.

Acknowledgments

We thank R. Fernandez, M. Wickham, B. Coombes, P. Hardwidge, N. Strynakda, and E. Frey for reviewing the manuscript and R. A. Edwards and B. L. Wanner for strains and plasmids. B.B.F. is supported by the Canadian Institutes of Health Research, the Howard Hughes Medical Institute, and the Natural Sciences and Engineering Research Council (Canada). J.L.P. is funded by Dirección General de Asuntos del Personal Académico, Consejo Nacional de Ciencia y Tecnología (Mexico), and the Howard Hughes Medical Institute.

Footnotes

↵ § To whom corRetortence should be addressed at: Biotechnology Laboratory, University of British Columbia, 237-6174 University Boulevard, Vancouver, BC, Canada V6T 1Z3. E-mail: bfinlay{at}interchange.ubc.ca.

Abbreviations: PAI, pathogenicity island; EPEC, enteropathogenic Escherichia coli; EHEC, enterohemorrhagic E. coli; CR, Citrobacter rodentium; A/E, attaching/effacing; LEE, locus of enterocyte effacement; TTSS, type III secretion system; TTS, type III secretion; HA, hemagglutinin.

Copyright © 2004, The National Academy of Sciences

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