Regulation of Expression and Secretion of NleH, a New Non-Locus of Enterocyte Effacement-Encoded Effector in Citrobacter rodentium

Abstract

Together with enterohemorrhagic Escherichia coli and enteropathogenic Escherichia coli, Citrobacter rodentium is a member of the attaching-and-effacing (A/E) family of bacterial pathogens. A/E pathogens use a type III secretion system (T3SS) to translocate an assortment of effector proteins, encoded both within and outside the locus of enterocyte effacement (LEE), into the colonized host cell, leading to the formation of A/E lesions and disease. Here we report the identification and characterization of a new non-LEE encoded effector, NleH, in C. rodentium. NleH is conserved among A/E pathogens and shares identity with OspG, a type III secreted effector protein in Shigella flexneri. Downstream of nleH, genes encoding homologues of the non-LEE-encoded effectors EspJ and NleG/NleI are found. NleH secretion and translocation into Caco-2 cells requires a functional T3SS and signals located at its amino-terminal domain. Transcription of nleH is not significantly reduced in mutants lacking the LEE-encoded regulators Ler and GrlA; however, NleH protein levels are highly reduced in these strains, as well as in escN and cesT mutants. Inactivation of Lon, but not of ClpP, protease restores NleH levels even in the absence of CesT. Our results indicate that the efficient engagement of NleH in active secretion is needed for its stability, thus establishing a posttranslational regulatory mechanism that coregulates NleH levels with the expression of LEE-encoded proteins. A C. rodentium nleH mutant shows a moderate defect during the colonization of C57BL/6 mice at early stages of infection.

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are important causes of infectious diarrhea. EPEC is the leading cause of severe watery bacterial diarrhea in children under 6 months of age living in developing countries. EHEC infections are associated with outbreaks of bloody diarrhea and hemorrhagic colitis in the developed world and can lead to severe kidney failure due to the development of the often-fatal hemolytic-uremic syndrome (33). EPEC and EHEC are noninvasive pathogens that infect their hosts by adhering to the surface of the intestinal epithelial cells. These organisms belong to a family of pathogens that are capable of producing a common histopathology called the attaching-and-effacing (A/E) lesion, which is characterized by the localized destruction of the enterocyte microvilli and the formation of pedestal-shaped actin-rich structures underneath the adherent bacteria (30). In vivo study of the mechanisms underlying EPEC and EHEC pathogenesis has been difficult, mainly due to the inability of these pathogens to efficiently colonize laboratory animals. Because of this, Citrobacter rodentium, an A/E pathogen that naturally colonizes laboratory mice, causing A/E lesions and disease, has emerged as a suitable model to study A/E infections (48).

The genes required for A/E lesion formation are located within the locus of enterocyte effacement (LEE) and encode an assortment of proteins involved in the assembly of a type III secretion system (T3SS), intimate attachment, effector functions, chaperones, and regulation, which to different degrees are all required for virulence (15, 21).

The LEE also encodes SepL and SepD, two proteins that form a molecular switch that confers specificity to the T3SS for the secretion of translocator proteins. In addition, this switch acts as a gatekeeper for the secretion of effector proteins, as sepD or sepL mutants hypersecrete them without secreting translocator proteins (14, 15). Proteomic analysis of the secreted proteins of a C. rodentium sepD mutant led us to the identification of seven new T3SS-secreted proteins, all of which are encoded outside the LEE in C. rodentium and other A/E pathogens. We named these proteins non-LEE-encoded effectors (Nle): NleA, NleB, NleC, NleD, NleE, NleF, and NleG (15). Further characterization of NleA (also called EspI), which is widely distributed among EHEC and EPEC strains (9, 10, 49), showed that it is critical for virulence and colonization of the mouse colon and upon translocation into epithelial cells localizes to the Golgi apparatus (27, 49). NleB, which plays a key role in C. rodentium virulence (35, 65), is encoded within O-island 122, which has been proposed to enhance virulence in EHEC O157:H7 strains (34), to be associated with diarrheal disease due to atypical EPEC (1), and to be associated with the LEE, forming a mosaic island in EHEC and atypical EPEC strains of serogroup O26 (4). It has also been demonstrated that NleC and NleD are translocated through the EHEC T3SS. NleC is not required for EHEC colonization of lambs or calves or for C. rodentium colonization of mice (35, 41). An EHEC EDL933 nleD mutant was originally identified by signature-tagged mutagenesis as being attenuated in calves (17); however, it was later reported that no attenuation was observed for nleD deletion mutants of both EHEC 85-170 and EDL933 (41). The nleE gene is also associated with EHEC O-island 122 (34), and C. rodentium nleE mutants are attenuated in mixed infections and produce delayed mortality in susceptible mice (35, 66). The role of NleG in vivo has not been studied; however, an NleG homologue found in EPEC, named NleI, has been shown to be translocated into epithelial cells in a T3S-dependent manner (39). A recent proteomic and bioinformatic approach expanded the number of effectors in EHEC to 39, which were classified into 20 families (61). In addition to the Nle proteins, other non-LEE-encoded effectors have been described in A/E pathogens (reviewed in reference 21).

Along with the acquisition of virulence factors, pathogenic bacteria have also acquired or developed regulatory mechanisms to ensure the appropriate spatio-temporal expression of such factors. In A/E pathogens, the expression of LEE-carried genes is tightly regulated by both global and A/E-specific regulatory factors (reviewed in reference 45). The LEE encodes three A/E-specific regulators. Ler (LEE-encoded regulator) has an essential role in the positive regulation of all the genes within the LEE (5, 28, 46, 54), as well as of others located outside the LEE such as espC in EPEC (20) and lpf in EHEC (62). Ler positively regulates LEE gene expression by counteracting the repression exerted by the global regulator H-NS (5, 28, 63). The first gene of the LEE1 operon encodes Ler, whose expression is negatively and positively regulated by a myriad of global regulatory proteins (reviewed in reference 45). In addition, A/E-specific regulatory proteins such as the EAF plasmid-encoded PerC in EPEC (5, 26, 50) or Pch in EHEC (32), as well as GrlA, which is also encoded within the LEE (3, 15), play critical roles in ler positive regulation, while GrlR, which is coexpressed with GrlA, has a negative role (15, 40). Interestingly, Ler and GrlA establish a positive regulatory loop that, together with GrlR, seems to modulate Ler levels in the cell (3). Ler and GrlA have been shown to play an essential role in vivo (15, 68). Furthermore, the regulatory mechanisms associated with the expression of effector genes located outside the LEE have just started to be elucidated (47, 51, 53).

Due to its association with the gene coding for the previously identified non-LEE-encoded effector NleF in C. rodentium and its similarity with OspG of Shigella flexneri, we hypothesized that the upstream gene, herein designated nleH, was likely to encode an effector protein. Using double-hemagglutinin (2HA)- and CyaA-tagged versions of NleH, we showed that its secretion and translocation to epithelial cells depend on a functional T3SS and that upon translocation it localizes to the cell membranes. Regulation of nleH was analyzed in different regulatory and secretory mutants by using an operon fusion between the entire nleH gene and the cat reporter gene. NleH is regulated at the posttranslational level by a novel mechanism that couples protein stability with Ler-GrlA-dependent regulation of LEE gene expression. Its role in colonization was assessed using the murine model of infection.

MATERIALS AND METHODS

Bacterial strains, plasmids, cell lines, and culture conditions.

The bacterial strains and plasmids used in this study are described in Table 1. Bacteria were grown at 37°C in Luria-Bertani (LB) broth or agar or in Dulbecco's modified Eagle's medium (DMEM). When required, the medium was supplemented with ampicillin (100 μg/ml) and kanamycin (30 μg/ml). Growth of C. rodentium in DMEM was carried out at 37°C in a 5% (vol/vol) CO₂ atmosphere without shaking. When EPEC was grown in DMEM, media were supplemented with 1% LB and incubated at 37°C with shaking (220 rpm). Caco-2 (ATCC HTB-37) cells were cultivated in minimum essential medium buffered with 2.4 g/liter HEPES and supplemented with 15% (vol/vol) fetal bovine serum and 200 mM l-glutamine at 37°C in a humidified 5% (vol/vol) CO₂ atmosphere.

TABLE 1.

Strains and plasmids used in this study

Strain, strain genotype, or plasmid	Description	Reference or source
C. rodentium strains
DBS100	WT (ATCC 51459)	55
Δler	DBS100 carrying an in-frame deletion of ler	15
ΔgrlA	DBS100 carrying an in-frame deletion of grlA	15
ΔgrlR	DBS100 carrying an in-frame deletion of grlR	15
ΔsepD	DBS100 carrying an in-frame deletion of sepD	15
ΔsepD ΔescN	DBS100 carrying an in-frame deletion of sepD and an in-frame deletion of escN	15
ΔescN	DBS100 carrying an in-frame deletion of escN	15
ΔcesT	DBS100 carrying an in-frame deletion of cesT	15
ΔnleH	DBS100 ΔnleH::Km	This study
ΔnleF	DBS100 ΔnleF::Km	66
EPEC strains
E2348/69	WT	38
ΔescN	E2348/69 carrying an in-frame deletion of escN	24
Δlon	E2348/69 carrying an in-frame deletion of lon	W. Deng, unpublished
ΔcesT Δlon	E2348/69 carrying an in-frame deletion of cesT and an in-frame deletion of lon	W. Deng, unpublished
ΔclpP	E2348/69 carrying an in-frame deletion of clpP	W. Deng, unpublished
ΔcesT ΔclpP	E2348/69 carrying an in-frame deletion of cesT and an in-frame deletion of clpP	W. Deng, unpublished
Δler	E2348/69 carrying an in-frame deletion of ler	V. H. Bustamante, unpublished
ΔgrlA	E2348/69 carrying an in-frame deletion of grlA	A. Huerta, unpublished
Δler Δlon	E2348/69 carrying an in-frame deletion of ler and an in-frame deletion of lon	This study
ΔgrlA Δlon	E2348/69 carrying an in-frame deletion of grlA and an in-frame deletion of lon	This study
Plasmids
pKD46	Red recombinase system under control of araB promoter; Apr	12
pKD4	Template plasmid containing the Km cassette for lambda Red recombinase	12
pTOPO-2HA	pCR2.1-TOPO derivative carrying C. rodentium espG coding region fused to two HA epitopes at the carboxyl terminus	15
pTnleH-HA	pTOPO-2HA derivative carrying C. rodentium nleH coding region fused to two HA epitopes at the caboxyl terminus	This study
pTnleH20-293HA	pTnleH-HA with nleH coding region deleted from codon 2 to 19	This study
pTnleH1-133HA	pTnleH-HA with nleH coding region deleted from codon 134 to 293	This study
pMPM-K3	Low-copy-no. cloning vector; p15A derivative; Kmr	44
pOG-ATCΔ304	pACYC184 derivative expressing amino acids 1-32 of Salmonella enterica SseK1 fused to CyaA	37
pK3cyaA	pMPM-K3 derivative containing cyaA residues 2-401	This study
pK3nleHcyaA	pK3cyaA containing nleH coding region fused to cyaA	This study
pK3nleH1-19cyaA	pK3nleHcyaA with nleH coding region deleted (codons 20-293)	This study
pKK232-8	pBR322 derivative containing a promoterless cat gene	Pharmacia LKB Biotechnology
pTregNleH-HA	pTOPO-2HA derivative containing C. rodentium nleH fused to two HA epitopes at the carboxyl terminus, including 1,749 bp of the regulatory region	This study
pKKnleH-HA	pKK232-8 derivative containing nleH-cat transcriptional fusion from nucleotide −1741 to +876 with respect to the start codon; nleH fused to two HA epitopes	This study
pKKespG-HA	pKKnleH-HA derivative containing espG-cat transcriptional fusion from nucleotide −363 to +1182 with respect to the start codon; espG fused to two HA epitopes	This study
pRE112	Suicide vector; sacB Cmr	18
pRE-Δler	Suicide construct carrying an in-frame deletion of EPEC ler in pRE112	V. H. Bustamante, unpublished
pRE-ΔgrlA	Suicide construct carrying an in frame deletion of EPEC grlA in pRE112	A. Huerta, unpublished

Construction of deletion mutants and plasmids.

Deletion of the nleH gene from C. rodentium DBS100 was performed by the one-step mutagenesis procedure developed by Datsenko and Wanner (12). The mutation eliminated the structural region of nleH and replaced it with a Km resistance marker. For the construction of plasmid pTnleH-HA expressing NleH tagged with a 2HA epitope, the DNA sequence containing the nleH putative ribosome binding site and the NleH-coding region without its stop codon was amplified by PCR from the C. rodentium DBS100 genome using primers NleHCF (5′-GCAAAAAGCTTCCGGTTTTTGTTGTCATGTCAGGG-3′) and NleHCR (5′-GCCACTCGAGAATTCTACTTAATACCACTCTGATAAG-3′) containing HindIII and XhoI sites, respectively. The resulting PCR product was digested with HindIII-XhoI and ligated into pTOPO-2HA (Table 1) digested with the same restriction enzymes. EPEC Δlon Δler and Δlon ΔgrlA mutants were generated by the sacB gene-based allelic exchange method as described previously (15) using suicide clones carrying in-frame deletions of grlA and ler (Table 1).

To generate pTnleH20-293HA and pTnleH1-133HA expressing truncated versions of NleH-2HA, pTnleH-HA DNA was used as the template for inverse PCR with primers RecHA (5′-AGCTCCGGTACCCATACATTTCAACCTTCAAAATAAGAC-3′) and RecH20B (5′-AGAAACGGTACCTCGCCTGATAATGCCGTCTTATCC-3′), which were designed to eliminate nleH codons 2 to 19 and to introduce KpnI sites. The resulting PCR products were digested with KpnI and self-ligated. To generate pTnleH1-133HA, pTnleH-HA DNA was used as the template for inverse PCR with primers NleHHAF (5′-AGTCGCTCGAGTATCCGTATGATG-3′) and NleH2AC160 (5′-ATTACCCTCGAGGGGTGACTTGTTATAGTCCAC-3′), which were designed to eliminate nleH codons 134 to 293 and to introduce XhoI sites. The resulting PCR products were digested with XhoI and self-ligated.

To generate a plasmid containing the 2HA-tagged nleH gene and the cat reporter gene forming an operon under the control of the nleH regulatory region, the nleH coding region without its stop codon plus 1,334 bp upstream of the translational start codon was amplified by PCR from the C. rodentium genome using primers NleHReg (5′-AAGTGCAAGCTTCCTCATAAGATGGAAAACTGC-3′) and NleHCR. The resulting PCR product was cloned into the HindIII-XhoI sites of pTOPO-2HA to generate pTregNleH-HA (Table 1). Next, PCRs were performed to amplify the whole insert from pTregNleH-HA, including the sequence corresponding to the 2HA epitope. The resulting PCR product was cloned into the BamHI-HindIII sites of pKK232-8 to generate pKKnleH-HA (Table 1). Likewise, pKKespG-HA was constructed using primers EspGHibF (5′-GTCCTAGGATCCCCGGGGCGGGGTCAGTCC-3′) and EspGHibR (5′-GTAATACTCGAGAGCATTGTTCAGATATGTTTCAG-3′), which were designed to amplify espG from the C. rodentium chromosome, including 353 bp upstream of the start codon. The PCR product was cloned into the BamHI-XhoI sites of pKKnleH-HA.

To create a vector suitable for the construction of CyaA reporter fusions, the sequence corresponding to codons 2 to 401 of the cyaA sequence contained in pOG-ATCΔ304 was amplified by PCR and cloned into the EcoRI-NotI sites of pMPM-K3, to generate pK3cyaA (Table 1). pK3cyaA possesses unique PstI and BglII sites at the cyaA 5′ end. To generate pK3nleHcyaA, the nleH coding region without its stop codon, plus 73 bp upstream of the start codon, was amplified from the C. rodentium chromosome and cloned into the HindIII-PstI sites of pK3cyaA. To construct pK3nleH1-19cyaA, pK3nleHcyaA DNA was used as the template for inverse PCR with primers containing PstI restriction sites that were designed to amplify nleH without codons 20 to 293. The resulting PCR product was PstI digested and self-ligated.

Protein secretion assays.

Fifty milliliters of DMEM with the appropriate antibiotic was inoculated with 500 μl of an LB broth overnight culture (optical density at 600 nm = 2) of the indicated strains, incubated for 1 h at 37°C in a 5% CO₂ atmosphere without shaking, and then induced with 0.5 mM or 5 μM IPTG (isopropyl-β-d-thiogalactopyranoside) and incubated for another 5 h. Bacteria were harvested by centrifugation at 17,900 × g for 10 min. Secreted proteins were precipitated from 1,350 μl of culture supernatants of each strain with 10% trichloroacetic acid at 4°C overnight and concentrated by centrifugation at 17,900 × g for 30 min at 4°C. The resulting fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis.

Cell fractionation.

Mechanical fractionation of infected epithelial cells was performed as previously described (23), with slight modifications. Caco-2 cell monolayers (approximately 2 × 10⁶ cells) in 75-cm² flasks were inoculated with 200 μl of a 3-h-preinduced DMEM culture of EPEC strains (multiplicity of infection = 25) carrying different plasmids. Infection proceeded for 1.5 h, and then the expression of NleH and its derivatives was induced by adding 0.5 mM IPTG and incubating for another 1.5 h. Media were discarded from monolayers, and cells were washed three times with phosphate-buffered saline (PBS) and harvested after treatment with 0.025% trypsin-0.2-g/liter EDTA in PBS. After the cells were washed three times with ice-cold PBS, they were resuspended in 300 μl of homogenization buffer (3 mM imidazole [pH 7.4], 250 mM sucrose, 0.5 mM EDTA) plus a protease inhibitor cocktail (Roche). Cells were mechanically disrupted by vigorous passage two times through a 22-gauge needle and six times through a 27-gauge needle. Low-speed centrifugation (3,000 × g) for 15 min was applied to this homogenate to pellet bacteria, unbroken Caco-2 cells, host nuclei, and cytoskeleton components (low-speed pellet). The supernatant was then subjected to ultracentrifugation at 33,000 × g for 60 min to separate the insoluble fraction (host cell membranes) from the soluble fraction (host cytosol). The resulting fractions were resolved by SDS-PAGE (10% polyacrylamide) and transferred to nitrocellulose for Western blot analysis.

Western blot assays.

Nitrocellulose membranes containing transferred proteins were blocked in 5% nonfat milk for 1 h. Membranes were incubated with either a 1:5,000 dilution of an anti-HA monoclonal (MAb) (Covance), 1:15,000 dilution of an anti-DnaK MAb (Invitrogen), a 1:5,000 dilution of an anti-CyaA MAb (Cedarlane), a 1:10,000 dilution of an anti-γ-tubulin MAb (Sigma), a 1:10,000 dilution of an anticalnexin rabbit polyclonal antibody (Sigma), or a 1:20,000 dilution of an anti-EspA rabbit polyclonal antibody (a kind gift of V. Sperandio and J. B. Kaper). Secondary antibodies included goat anti-rabbit immunoglobulin G (Pierce) at a 1:10,000 dilution and rabbit anti-mouse immunoglobulin G (Pierce) at a 1:10,000 dilution. Positive signals were visualized with Western Lightning Chemiluminescence Reagent Plus (Perkin-Elmer).

Infection analysis of C. rodentium in mice.

Three- to 4-week-old C57BL/6 mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Wild-type (WT) C. rodentium and its nleH mutant derivative were grown in LB broth overnight at 37°C at 200 rpm. Mice were inoculated by oral gavage with 100 μl of these cultures. The inoculum was titrated by serial dilution and plating and was calculated to be approximately 2.5 × 10⁸ CFU per mouse for all groups. To assay bacterial colonization, mice were sacrificed and the first 4 cm of the distal colon starting from the anal verge was collected. Colonic tissue and fecal pellets were homogenized using a Polytron tissue homogenizer and serially diluted before being plated on MacConkey agar (Difco Laboratories) to determine the total bacterial burden in the mouse at the time of sacrifice.

CAT assay.

Determination of chloramphenicol acetyltransferase (CAT) activity and protein quantification to calculate CAT specific activities were performed as previously described (42).

Statistical analysis.

One-way analysis of variance, using SPSS software, was used for statistical analysis.

RESULTS

Identification of nleH in the genome of C. rodentium.

We previously reported the identification, by proteomic analysis, of a new set of putative effector proteins secreted by a sepD mutant strain of C. rodentium in a T3S-dependent manner (15). Genome sequence analysis revealed that these proteins were encoded not by genes within the LEE but by genes scattered throughout the genome that were located in cryptic prophages or putative pathogenicity islands, and they therefore were designated non-LEE-encoded effectors (Nle) (15). Analysis of the chromosomal location of nle genes in the partially sequenced C. rodentium genome (http://www.sanger.ac.uk) showed that nleF and a nleG homologue were located in a chromosomal locus inserted between the yjjG and prfC genes, which are contiguous in the EHEC EDL933 and nonpathogenic E. coli K-12 chromosomes (Fig. 1A). This locus spans 12.6 kb and also contains the gene coding for the T3SS-translocated effector EspJ, which is found in a different location in EHEC and EPEC (11). Besides the described effector genes, in this locus we found an open reading frame of 293 codons immediately upstream of nleF, whose product shares 15% identity with OspG, an effector of Shigella flexneri (36) (Fig. 1B). Because of its location in the C. rodentium genome and the fact that its predicted amino acid sequence shares identity with a type III secreted effector in S. flexneri, we speculated that this gene codes for an effector protein, herein named NleH. In addition, we had also found that an NleH homologue present in the secreted proteins of EPEC ΔsepD interacts with CesT (60).

FIG. 1.

NleH is conserved in A/E pathogens. (A) Schematic representation of the loci containing nleH in C. rodentium DBS100 and the two homologues of nleH in EPEC E2348/69 and EHEC EDL933. NleH1 and NleH2 correspond, respectively, to Z6021 and Z0989 in the EHEC EDL933 genome sequence, to ECs1814 and ECs0848 in the EHEC Sakai genome sequence, and to NleH1-2 and NleH1-1 according to a recently suggested nomenclature (64). Flanking genes correspond to those conserved in E. coli K-12 and are considered the boundaries of the insertion sites of this putative horizontally acquired set of genes. (B) Multiple alignment of NleH homologues from C. rodentium, EPEC E2348/69, and EHEC EDL933 and OspG from Shigella flexneri M90T. Sequences of C. rodentium and EPEC E2348/69 were obtained from the sequencing genome project at the Sanger Institute (www.sanger.ac.uk). Conserved amino acid residues involved in NF-κB signaling as described for OspG (36) are indicated by arrowheads.

NleH shares a high degree of identity (83%) with the predicted protein products of two open reading frames of unknown function found in the genome of EHEC EDL933, designated Z6021 and Z0989, herein called NleH1 and NleH2, respectively (Fig. 1B). In the partially sequenced genome of EPEC strain E2348/69 (http://www.sanger.ac.uk), we also found two NleH homologues sharing around 83% identity with C. rodentium NleH. The loci containing the EPEC and EHEC nleH homologues do not share the chromosomal insertion site with C. rodentium nleH. The nleH1 gene in EHEC is part of a 58.6-kb chromosomal locus containing prophage genes and the genes coding for NleA, NleF, and a NleG-like protein, which interrupt yciD, between E. coli backbone genes yciE and ynfA. In EPEC, the 55.5-kb locus containing nleH1 is inserted between yciD and trpA and also contains nleA and nleF but not nleG (Fig. 1A). nleH2 genes in EPEC and EHEC are also found in regions containing prophage structural genes. In both cases the region containing nleH2 is inserted between the E. coli K-12 backbone genes ybhC and ybhB, though the size of the locus in each case is different (Fig. 1A). In EPEC the nleH2 locus spans 52.3 kb and in EHEC 38.1 kb. In EPEC, the nleH2 locus contains a cif homologue, while in EHEC the nleH2 locus contains homologues of three nle genes: nleD, nleC, and nleB2. The gene organization of the nleH locus in C. rodentium is similar to that of the nleH1 loci of both EHEC and EPEC; however, the predicted protein shares higher identity with NleH2, including the lack of a 10-amino-acid indel between residues 29 and 30 (Fig. 1B).

NleH is a substrate of the LEE-encoded T3SS.

To test whether NleH is a substrate of the T3SS in C. rodentium, plasmid pTnleH-HA expressing NleH-2HA (Fig. 2A) was transformed into WT C. rodentium and its T3S-deficient ΔescN and effector-hypersecreting ΔsepD derivatives. These strains were grown in DMEM at 37°C for 6 h in the presence of 100 μM IPTG to induce the expression of NleH-2HA. The secreted proteins were recovered from culture supernatants by trichloroacetic acid precipitation and subjected to Western blot analysis using anti-HA antibodies (Fig. 2B). NleH-2HA was expressed in all the strains tested but was detected only in the culture supernatants of the WT and ΔsepD strains, as expected for a T3S effector (Fig. 2B). Together, these results demonstrate that NleH is secreted through the LEE-encoded T3SS in C. rodentium.

FIG. 2.

NleH is secreted through the LEE-encoded T3SS. (A) Schematic representation of the NleH-2HA and NleH-CyaA reporter fusions and deletion derivatives used in this study. (B) Western blot assay of total and secreted proteins of C. rodentium WT, T3SS-deficient ΔescN, and effector hypersecretion ΔsepD strains expressing NleH-HA from pTnleH-HA. Samples were separated by SDS-PAGE and probed with anti-HA antibodies. Blots were also probed with anti-DnaK antibodies as a loading control and to monitor bacterial lysis in the secreted protein fractions. (C) The first 19 amino acid residues of NleH are sufficient to mediate T3S. Bacterial pellets and secreted proteins of WT C. rodentium harboring plasmids pTnleH-HA, pTnleHΔ2-19HA, and pTnleHΔ134-293HA were analyzed by Western blotting with anti-HA and anti-DnaK antibodies. (D) Plasmids expressing NleH-CyaA (pK3nleHcyaA) and NleH_(1-19)-CyaA were transformed into C. rodentium WT, ΔescN, and ΔsepD strains. Samples were collected from DMEM cultures. Whole-cell extracts and secreted proteins were separated by SDS-PAGE and analyzed by Western blotting using anti-CyaA and anti-DnaK antibodies.

Although a signature secretion signal has not been described for T3S effectors, it has been determined to reside within the first 10 to 20 N-terminal amino acid residues (8, 58). To determine whether the NleH secretion signal was located at the N or C terminus, two NleH-2HA derivatives were generated by deleting codons 2 to 19 and 134 to 293, obtaining plasmids pTnleH20-293-HA and pTnleH1-133HA, respectively (Fig. 2A). The secretion of these NleH derivatives was tested in C. rodentium, where, as shown in Fig. 2C, the construct lacking the first 19 amino acids was expressed but not detected in culture supernatants. In contrast, deletion of the C terminus did not affect expression or secretion, indicating that, as for other T3S effectors, the signal required for NleH secretion resides at the amino-terminal domain.

To further support this observation, two CyaA reporter fusions were constructed, with the entire NleH amino acid sequence (pK3nleHcyaA) and with only the first 19 amino-terminal residues (pK3nleH1-19cyaA) (Fig. 2A). In accordance with our previous results, the complete NleH-CyaA fusion was secreted by WT C. rodentium, as it was detected by Western blotting with anti-CyaA antibodies in the culture supernatant (Fig. 2D). The presence of two NleH-CyaA processing products indicated that this construct was somehow unstable, but it did not interfere with the secretion of the entire fusion. The CyaA fusion containing NleH residues 2 to 19 was detected in the culture supernatants of the WT and ΔsepD strains but not in the supernatant of the ΔescN strain, demonstrating that the first 19 residues of NleH are sufficient to confer type III-dependent secretion (Fig. 2D).

NleH associates with host cell membranes upon translocation.

In order to determine whether NleH is also translocated into epithelial cells through the LEE-encoded T3SS, we used EPEC E2348/69 and its ΔescN derivative expressing the NleH-2HA and NleH-CyaA fusions depicted in Fig. 2A to infect Caco-2 cells and perform fractionation assays. It has been shown that EPEC adheres more efficiently to epithelial cells in vitro, thus providing a more sensitive way to evaluate the T3SS-dependent translocation of effector proteins by A/E pathogens into epithelial cells (11, 41, 64). Infected cells were subjected to mechanical fractionation and the fractions obtained analyzed by Western blotting using anti-HA and anti-CyaA antibodies (Fig. 3). These results showed that upon translocation, full-length NleH-2HA and NleH-CyaA associate mainly with the host membrane fraction and that this association requires a functional T3SS, as NleH was not found in membrane fractions of cells infected with EPEC ΔescN (Fig. 3). NleH_(1-133)-HA was also found to be translocated and associated with the membrane fraction, indicating that the carboxyl terminus does not contain the translocation or membrane association signal. In contrast, NleH_(1-19)-CyaA was not translocated, suggesting that even when sufficient for secretion, the first 19 amino acids are not enough for translocation. Collectively, these results indicate that while secretion is achieved with only the first 19 amino acids, the signals sustaining translocation and membrane association of NleH are located within its first 133 amino acids.

FIG. 3.

NleH translocation into Caco-2 cells. Caco-2 cells were infected with WT and ΔescN EPEC E2348/69 strains harboring plasmids expressing the NleH fusions described in Fig. 2A. Upon IPTG induction, cells were subjected to fractionation by mechanical lysis as described in Materials and Methods. The low-speed pellet fraction contains intact Caco-2 cells, complete organelles, cytoskeleton components, and intact bacteria. As fractionation controls, all blots were analyzed using anti-HA, anti-CyaA, anti-DnaK, anticalnexin, and antitubulin antibodies. Each fractionation was done twice with identical results.

Regulation of NleH expression by LEE-encoded regulators.

Despite the fact that the number of identified non-LEE-encoded effectors in A/E pathogens has grown significantly in recent years, little is known about their regulation. As NleH is secreted and translocated in a T3S-dependent manner, we investigated whether its expression is coregulated with the expression of LEE-encoded genes. To monitor the expression of both the protein and a reporter gene under the control of the nleH regulatory region, we constructed an operon fusion between the nleH regulatory and structural regions double tagged with the HA epitope and the CAT gene in plasmid pKK232-8, generating plasmid pKKnleH-HA (Fig. 4A). To assess the role of LEE-encoded regulators in NleH expression, we introduced pKKnleH-HA in C. rodentium WT, Δler, ΔgrlA, and ΔgrlR strains. Strains were grown in DMEM at 37°C without shaking under a 5% CO₂ atmosphere for 6 h, which are conditions known to induce LEE gene expression in C. rodentium (3, 15). Culture samples were used to determine CAT activity and analyze NleH-2HA expression and secretion by Western blotting. As shown in Fig. 4B, the CAT activity directed by the fusion contained in pKKnleH-HA was not significantly reduced in the Δler and ΔgrlA strains with respect to the WT strain, suggesting that nleH transcription is not part of the Ler-GrlA regulon. In contrast, NleH-HA was undetectable by Western blotting in both whole cells and culture supernatants of the Δler and ΔgrlA mutants (Fig. 4C). Although not statistically significant, the CAT activities directed by the fusion contained in pKKnleH-HA showed opposite trends in the grlA (reduced expression) and grlR (enhanced expression) mutants, suggesting that GrlA and GrlR may modulate NleH expression positively and negatively, respectively. This possibility is currently being investigated. As a control we constructed a fusion similar to pKKnleH-HA for the LEE-encoded effector EspG, whose expression is regulated by Ler (45), generating pKKespG-HA (Fig. 4D). As expected, CAT activity and EspG-2HA expression were markedly reduced in the Δler and ΔgrlA strains carrying pKKespG-HA (Fig. 4E and F).

FIG. 4.

Regulation of NleH. (A) Schematic representation of the transcriptional fusion of nleH to the cat reporter gene (pKKnleH-HA). The structural region of nleH marked with a 2HA epitope tag was fused as an operon to the promoterless cat reporter gene. The fusion includes 1,741 bp upstream of the nleH start codon. (B) CAT specific activity driven by pKKnleH in the indicated C. rodentium strains after 6 hours of growth in DMEM cultures. Results shown are the averages and standard deviations from four independent experiments. (C) Western blot assay of whole-cell extracts and secreted proteins of the strains used in the experiment shown in panel B, using anti-HA, anti-EspA, and anti-DnaK antibodies. An experiment with representative results is shown. (D) Schematic representation of the transcriptional fusion of espG to the cat reporter gene (pKKespG-HA). The structural region of espG marked with a 2HA epitope tag was fused in an operon to cat. The fusion includes 353 bp upstream of the espG start codon. (E) CAT specific activity obtained from culture samples of strains carrying pKKespG-HA after 6 hours of growth in DMEM. Results shown are the averages and standard deviations from four independent experiments. (F) Western blot analysis of whole-cell extracts and secreted proteins of the strains shown in panel E. An experiment with representative results is shown.

NleH is not stable in the absence of CesT or active secretion.

The results described above suggested that NleH levels are regulated posttranscriptionally, in a Ler- and GrlA-dependent manner, probably by controlling the expression of LEE-encoded proteins important for NleH stability and secretion. In accordance to this, we have recently reported that NleH is a binding substrate of CesT and that in its absence NleH becomes unstable (59). CesT is encoded by the LEE5 operon, whose expression is regulated by Ler (54). We have also shown that CesT interacts with the T3SS membrane-associated ATPase EscN (60), which is encoded by the Ler-dependent operon LEE3 (5). To further test this possibility, plasmids pKKnleH-HA and pKKespG-HA were transformed into ΔcesT, ΔescN, and ΔsepD ΔescN mutant strains, all of which are deficient in effector secretion (15, 60), as well as in a ΔsepD strain. As expected, NleH was not secreted by or present in whole cells of the ΔcesT mutant, and the ΔescN mutant showed a very similar phenotype, while the ΔsepD mutant showed higher levels of secretion (Fig. 4C). Moreover, the higher levels of NleH observed in a ΔsepD mutant, both in the secreted fraction and whole cells, were drastically reduced in a ΔsepD ΔescN double mutant (Fig. 4C). In all cases, reduced NleH levels were not due to an effect on transcription (Fig. 4B). Taking together, these results indicated that the absence of NleH in the ler and grlA regulatory mutants (Fig. 4C) is most likely due to the lack of expression of CesT, EscN, and T3SS components in general and thus to the lack of secretion, which seems to make NleH accessible to degradation. To further confirm that NleH is not stable in the Δler and ΔgrlA regulatory mutants, these strains were transformed with pTnleH-HA, which expresses NleH-2HA from an IPTG-inducible promoter (Table 1). In contrast to the case for the WT and ΔgrlR strains, NleH-2HA was not seen in whole cells or culture supernatants of the Δler and ΔgrlA strains (Fig. 5A). Together, these results indicated that even when NleH was expressed from an inducible promoter, its stability was dependent not only on the presence of its chaperone but also on active secretion. Interestingly, the EspG effector protein was not subjected to the same posttranslational control, as EspG-2HA could be detected in whole cells of all strains, except for the Δler and ΔgrlA strains, and as a secreted protein in the ΔcesT strain when expressed from its own promoter (Fig. 4F). When expressed from an inducible promoter, EspG-2HA could be detected even in whole cells of the regulatory mutants but not, as expected, in their secreted proteins (Fig. 5B).

FIG. 5.

NleH expressed from P_lac is coordinately regulated with the LEE-encoded T3SS. Western blot assays of the bacterial pellet and secreted proteins of the indicated C. rodentium strains carrying pTnleH-HA expressing NleH-HA from P_lac (A) and pTOPO-2HA expressing EspG-HA from P_lac (B), using anti-HA and anti-DnaK antibodies, are shown. Expression was induced with 5 μM IPTG for 5 h. Assays were done twice with identical results.

Lon, but not ClpP, is involved in the degradation of NleH in the absence of CesT.

Energy-dependent proteases such as Lon and ClpP have been involved in the expression or stability of T3S effectors in different pathogenic bacteria (reviewed in reference 6). To explore whether these proteases are responsible for the rapid turnover of NleH observed in the absence of CesT, NleH-2HA was expressed in WT EPEC and its ΔcesT, ΔcesT Δlon, and ΔcesT ΔclpP derivatives. As shown in Fig. 6A, NleH-2HA was readily detected in whole cells of the ΔcesT strain in the absence of the Lon protease but was still degraded in the absence of ClpP, suggesting that when NleH is not being recruited by CesT and actively secreted, this effector protein is being specifically targeted for degradation by Lon. We then addressed whether the lon mutation was able to restore NleH stability in the Δler and ΔgrlA backgrounds. Interestingly, NleH stability was restored in the ΔgrlA Δlon but not in the Δler Δlon double mutant (Fig. 6A), revealing that the posttranslational regulation of NleH may involve two different pathways and that more than one Ler-regulated component, in addition to CesT, may be involved in NleH protein stability. This result prompted an additional experiment in which the roles in protein instability of the N- and C-terminal domains of NleH were tested by expressing the N-terminal [NleH_(20-293)-HA] and C-terminal [NleH_(1-133)-HA] deletions in Δler and ΔgrlA mutants. Consistent with the notion that two different pathways are involved in NleH degradation, only the N-terminal deletion was stable in the ΔgrlA mutant, while none of the NleH deletion derivatives was stable in the Δler mutant (Fig. 6B). These observations are in line with our recent observation that the CesT binding domain of NleH is contained within the first 40 amino acids (59).

FIG. 6.

Lon protease degrades NleH in the absence of CesT. (A) Western blot assay of bacterial pellets of EPEC strains (as indicated) transformed with pTnleH-HA, which expresses an NleH-HA fusion from P_lac, using anti-HA and anti-DnaK antibodies. Expression of the fusion was induced with 5 μM IPTG for 5 h. (B) Analysis of the stability of full-length NleH-HA and of its amino- and carboxy-terminal deletions NleH_(20-293)-HA and NleH_(1-133)-HA (illustrated in Fig. 2A). Western blot assays of WT, Δler, and ΔgrlA strains of C. rodentium containing pTnleH-HA, pTnleH20-293HA, and pTnleH1-133HA plasmids are shown. Strains were grown in DMEM, and the expression of fusions was induced with 5 μM IPTG for 5 h. Assays were done twice with identical results.

Lack of NleH delays colonization of C57BL/6 mice by C. rodentium.

Considering that NleH displays different features of a T3S effector, its role as a virulence factor for C. rodentium was assessed by infecting C57BL/6 mice with C. rodentium DBS100 and its nleH null mutant. Infected animals were sacrificed at days 6 and 10 postinfection (p.i.), and the total bacterial burden in the mouse colon was calculated. The nleH mutant showed a moderate, statistically significant (P = 0.01) reduction in its capacity to colonize the mouse colon at day 6 p.i. (Fig. 7A). However, no difference in colonization between the WT and ΔnleH strains was observed at day 10 p.i. (Fig. 7B). In contrast, a strong attenuation was observed for the nleB and escN mutants (data not shown), as previously reported (15, 35, 65). Overall, these results indicate that NleH has a role in promoting efficient colonization of the mouse colon by C. rodentium during early stages of infection.

FIG. 7.

NleH is required at early stages during colonization in the mouse model of infection. Groups of C57BL/6 mice were orally infected with equal numbers of WT or ΔnleH bacteria. Mice were sacrificed at day 6 (A) or 10 (B) p.i., and the total burden of C. rodentium in the distal colon was assessed by MacConkey serial dilution platting. *, statistically significant difference with respect to the WT strain (P = 0.01).

DISCUSSION

In recent years, the development of effector hypersecretion mutants combined with the use of proteomics and bioinformatics has allowed the identification of a significant number of new T3S effector proteins in A/E pathogens, which are encoded outside the LEE pathogenicity island (15, 61). In this work, we describe different features of NleH, a non-LEE-encoded effector that we originally identified in C. rodentium based on its homology to S. flexneri OspG and its genetic linkage to other previously characterized T3S effectors (Fig. 1A) (15), as well as in EPEC as a CesT binding substrate (60).

In C. rodentium, nleH is located in a 12.6-kb locus inserted between the yjjG and prfC genes that has no apparent structural phage genes. In EPEC E2348/69 and EHEC EDL933 there are two loci carrying nleH homologues (nleH1 and nleH2), but their site of insertion is different with respect to that in C. rodentium (Fig. 1A). The fact that both nleH copies in EHEC and EPEC are prophage carried (61) is not unexpected given the importance that phages have acquired as virulence gene dissemination vectors, in particular T3S effectors (19, 61). Furthermore, for EPEC serogroups O86a, O127, and O142, isolated from patients with acute diarrhea, it has recently been reported that the locus carrying nleH2 is an inducible lambdoid prophage that also encodes the cytolethal-distending toxin (Cdt-I) (2), an observation that highlights the potential mobility of these putative virulence factors. Differences in the genomic context of the nleH homologues, as for other T3SS effectors, support the notion that the acquisition of effector genes by A/E pathogens has been the result of multiple and independent horizontal transfer events.

Using NleH-2HA- and NleH-CyaA-tagged proteins, we showed that the first 19 N-terminal amino acid residues of C. rodentium NleH are essential and sufficient for its secretion through the LEE-encoded T3SS but not for its translocation into epithelial cells. In contrast, it has been reported that the first 20 amino acids are sufficient to mediate both secretion and translocation of other A/E effectors, such as Tir, EspF, Map, Cif, and NleA/EspI (8). However, our results are consistent with the notion that the secretion signal resides within the first 15 to 20 amino acids of effector proteins of different T3SSs, while translocation signals are located further down the N-terminal domain (25). In fact, the modular organization of N termini consisting of separated secretion and translocation domains has been reported for other effectors, such as Yersinia YopE and YopH (57). Secretion and translocation of NleH homologues in EPEC and EHEC have also been recently reported (59, 61). In addition, we show that upon translocation NleH associates with the membrane fraction, likely corresponding to the cytoplasmic membrane instead of organelle membranes, considering that upon translocation EPEC NleH localizes underneath adherent bacteria, as we have recently reported (59).

Another distinctive feature of all LEE-encoded effectors, as well as of T3SS components, is that their expression is coordinated at the transcriptional level by Ler (45). As non-LEE-encoded effectors are secreted by the LEE-encoded T3SS, it has been tempting to speculate that their expression is coregulated with the LEE. In this regard, it has been recently reported that nleA, nleB, nleD, and nleE are transcribed in EHEC under secretion-permissive conditions and that the expression of nleA::gfp, nleA::bla, and nleD::bla translational reporter fusions showed a reduction in the absence of Ler (53). This result, however, did not distinguish whether Ler-mediated regulation occurs at the transcriptional or posttranslational level. Interestingly, we found here that even when Ler and GrlA do not seem to play a significant role in nleH transcription, the Ler-mediated regulation of LEE expression influences posttranslationally the expression of NleH, as even when NleH was expressed from an inducible promoter, its levels in the cell were drastically reduced in the absence of these regulatory proteins. In addition, NleH protein levels were greatly diminished in the absence of CesT, as well as in secretion-deficient strains such as the escN mutant, although no significant effects were seen for nleH transcription in these strains.

We have recently shown that CesT is a multieffector chaperone that recruits different LEE- and non-LEE-encoded effectors for their efficient T3S (59, 60). These observations led to us to speculate that when NleH in not actively engaged for secretion, its protein levels may be controlled by the competing action of proteases to prevent its potentially toxic accumulation within the cell. One would expect that it would be more energetically favorable to regulate the expression of these non-LEE-encoded effector proteins at the transcriptional level, through the same mechanisms that regulate the expression of LEE genes. However, we still have to determine when and under which conditions NleH is expressed, as we cannot rule out the possibility that the spatio-temporal expression of NleH differs from that of LEE-encoded genes. This would imply that its Ler-independent transcriptional regulation is required to ensure its expression at a different stage of the infection or at a different niche, once the T3SS components are all in place. Thus, if T3S does not occur, NleH expression would be posttranscriptionally regulated, by controlling either mRNA stability, translation, or protein stability. The first two options do not seem to be the case, as no polar effect is seen in the expression of the cat reporter gene when in an operon with nleH.

Regarding protein stability, recent reports have established that energy-dependent proteases, such as Lon and ClpP, control the intracellular concentration of effector proteins in different bacterial pathogens expressing T3SS effectors (reviewed in reference 6). Here, we show that NleH is fully stable in the absence of CesT when Lon, but not ClpP, is inactive, suggesting that when NleH is not bound by CesT and/or engaged for secretion, Lon-specific proteolysis degrades the accumulated protein to prevent undesired effects. Interestingly, the mutation of lon restored NleH levels in the ΔgrlA mutant but not in the Δler mutant, suggesting that NleH stability may rely on, in addition to CesT, another NleH-stabilizing factor regulated by Ler that prevents NleH degradation by a Lon-independent pathway. Consistently, we also observed differential stability between the N-terminal and C-terminal deletion derivatives of NleH in the Δler and ΔgrlA mutants. In line with these observations, even in the presence of CesT, lack of secretion of NleH in the ΔescN mutant also prompts its degradation, while higher levels of NleH are secreted when actively engaged in secretion in the ΔsepD hypersecretion mutant. Thus, in the absence of GrlA, although the expression of Ler is not completely abolished, it is nevertheless highly reduced (3, 15). This low level of Ler expression may not support the expression of CesT to sustain NleH stability but may be enough to activate the expression of an additional NleH-stabilizing factor that is needed to prevent NleH degradation from the C terminus. This model may explain why the stability of NleH and its derivatives was not restored in the ΔlerΔlon mutant, where the lack of Ler has a profound effect on the expression of the Ler regulon (reviewed in reference 45). In this way, under environmental conditions that are not permissive for Ler-dependent expression of LEE genes, the lack of CesT, as well as of T3SS and other Ler-regulated elements, will prompt the degradation of NleH by endogenous proteases and probably the degradation of other non-LEE-encoded effectors, whose expression is not directly controlled by Ler. Of note, ClpP has instead been shown to play a positive role in controlling the expression of LEE genes by regulating RpoS and GrlR levels in EHEC (31). However, the slight increase of nleH transcription seen in the ΔgrlR strain does not seem to account for the higher levels of accumulated and secreted NleH seen in the C. rodentium grlR mutant. This suggests that GrlR could also be linked to the posttranslational control of NleH expression, since when expressed under the control of an inducible promoter, higher levels of NleH are still observed in the ΔgrlR mutant than in the WT. It is interesting to note that this putative posttranslational control does not apply for the Ler-regulated LEE-encoded effector EspG, whose protein levels are not reduced in regulatory mutants when expressed from an inducible promoter.

Overall, these observations confirmed that NleH behaves as a T3S effector and prompted the experiments to address its potential role during infection. Initial experiments to look at pedestal formation and actin accumulation underneath adherent bacteria on epithelial cells revealed that A/E lesion formation was not affected in the absence of NleH (data not shown). This was not completely unexpected, as in addition to Tir only one effector encoded outside the LEE, EspFu/TccP, has been shown to be required for pedestal formation (7, 22). We then tested the ability of the C. rodentium nleH mutant to colonize C57BL/6 mice, which have become an accepted model for the study of the disease caused by A/E pathogens (48). In this assay, the nleH mutant showed a moderate, but statistically significant, reduction of about 1 log in its ability to colonize the mouse colon at day 6 p.i.; however, this defect was overcome at day 10 p.i. This result suggests that NleH may play a role at early stages of infection by C. rodentium, as has been suggested for the LEE-encoded effector EspG (29). It is worth noticing that among all the effectors described to date, only Tir, EspZ/SepZ, NleA/EspI, and NleB have been shown to play a critical role in the colonization of the mouse colon by C. rodentium (15, 16, 27, 35, 49, 65). The other tested effector proteins have been shown to have only moderate effects on colonization or on the outcome of the disease (see the introduction). Considering the diverse distribution of genetic variability that is being observed among different human and animal isolates of EHEC O157:H7, non-O157 Shiga toxin-producing E. coli, EPEC, and atypical EPEC strains, it is tempting to speculate that the potential redundancy between different effector proteins, due either to the existence of genes encoding highly similar proteins, as in the case of NleH in EPEC and EHEC, or to functional mimicry despite not sharing sequence similarity, may obscure the roles of some of them during infection.

Functional redundancy has been reported for EspG and EspG2, two effector proteins that share 43.5% identity in EPEC. Both EspG and EspG2 can independently activate RhoA in the host cell and increase paracellular permeability (43), besides being able to induce microtubule elimination (56). Map and EspF, two effector proteins that are not related at the sequence level, participate in EPEC-mediated disruption of epithelial barrier function, which can occur in the absence of Map or EspF but completely disappears in a map espF double mutant (13). Furthermore, the results obtained during the in vivo evaluation of effector gene mutants may vary depending on the animal model employed. For example, a C. rodentium espH mutant colonizes and causes hyperplasia as well as the WT strain in mice (49); however, the inactivation of espH in EHEC affects its capacity to colonize the intestinal tracts of baby rabbits and reduces the severity of diarrhea caused in this model (52). Due to differences among A/E pathogens and specificity for their hosts, the analysis of different models may be necessary to identify the function of a particular effector protein. Moreover, some effectors have been more frequently associated with A/E strains derived from severe cases of the disease. For example, NleE, which plays a moderate role in colonization and hyperplasia in the mouse model of infection (35, 66), has been linked to diarrhea production by atypical EPEC strains (1) and to hemolytic-uremic syndrome development and outbreaks by non-O157 EHEC strains (65). In a recent work, a microarray-based comparative genomic hybridization technique was used to detect differences in the genomic contents of 31 0157:H7 EHEC strains. Interestingly, the presence of nleH1, nleH2, and nleF was found to be associated with lineage I strains, which are more frequently linked to human disease (67).

The role in disease of the ample collection of Nle proteins, either individually or in concert, in A/E pathogens may depend on several, but at least two, different aspects: the genetic repertoire of each strain and the host's characteristics. These elements may influence the pathogenic potential of the strains, including their ability to colonize different hosts. In addition, A/E pathogens may have implemented different mechanisms to coordinate and control the expression of these effector proteins. The tremendous challenge we face in future studies is to elucidate the molecular mechanisms underlying the coordinated expression and function in pathogenesis and disease of such an extended repertoire of effector proteins.