A Distinct Regulatory Sequence Is Essential for the Expression of a Subset of nle Genes in Attaching and Effacing Escherichia coli

Abstract

Enteropathogenic Escherichia coli uses a type III secretion system (T3SS), encoded in the locus of enterocyte effacement (LEE) pathogenicity island, to translocate a wide repertoire of effector proteins into the host cell in order to subvert cell signaling cascades and promote bacterial colonization and survival. Genes encoding type III-secreted effectors are located in the LEE and scattered throughout the chromosome. While LEE gene regulation is better understood, the conditions and factors involved in the expression of effectors encoded outside the LEE are just starting to be elucidated. Here, we identified a highly conserved sequence containing a 13-bp inverted repeat (IR), located upstream of a subset of genes coding for different non-LEE-encoded effectors in A/E pathogens. Site-directed mutagenesis and deletion analysis of the nleH1 and nleB2 regulatory regions revealed that this IR is essential for the transcriptional activation of both genes. Growth conditions that favor the expression of LEE genes also facilitate the activation of nleH1 and nleB2; however, their expression is independent of the LEE-encoded positive regulators Ler and GrlA but is repressed by GrlR and the global regulator H-NS. In contrast, GrlA and Ler are required for nleA expression, while H-NS silences it. Consistent with their role in the regulation of nleA, purified Ler and H-NS bound to the regulatory region of nleA upstream of its promoter. This work shows that at least two modes of regulation control the expression of effector genes in attaching and effacing (A/E) pathogens, suggesting that a subset of effector functions may be coordinately expressed in a particular niche or time during infection.

INTRODUCTION

Enteropathogenic Escherichia coli (EPEC) is the leading cause of severe watery diarrhea in children in developing countries. This Gram-negative bacterium belongs to the attaching and effacing (A/E) family of pathogens, which includes enterohemorrhagic E. coli (EHEC) and the murine pathogen Citrobacter rodentium. A/E pathogens colonize the intestinal tract of their host and use a type III secretion system (T3SS) to translocate an assortment of proteins called effectors into the cytosol of the enterocyte which subvert cell signaling pathways in order to promote bacterial colonization and proliferation. The characteristic A/E lesion caused by these pathogens consists of epithelial microvillus depletion and the formation of actin-rich pedestals beneath adhering bacteria (reviewed in references 17, 33, 45, 67, and 93).

The genes required for assembly of the T3SS in A/E pathogens are encoded by the locus of enterocyte effacement (LEE) pathogenicity island. Besides the structural T3SS genes, the LEE also encodes regulatory proteins, effectors, chaperones, and proteins involved in the hierarchical regulation of secretion (18, 29). Substrate effectors of the T3SS are encoded in the LEE, as well as in prophages and integrative elements scattered throughout the chromosome. LEE-encoded effectors include Tir, EspF, Map, EspH, EspG, and EspZ. These effectors are highly conserved in A/E pathogens and have been shown to promote bacterial adhesion and actin and tubulin rearrangements and to alter epithelial barrier function and mitochondrial membrane potential, among other cellular events (reviewed in references 8, 15, and 29). However, the repertoire of non-LEE-encoded effectors (Nle) is variable in A/E pathogens. Genome sequence analysis has shown that EPEC strain E2348/69, EHEC strain Sakai, and C. rodentium have a repertoire of 21, 50, and 29 genes (excluding pseudogenes), respectively, encoding confirmed and putative type III-secreted effectors (T3SE) (38, 74, 89). Secretion and translocation of most of these effectors have been confirmed using biochemical and proteomic approaches (17, 19, 89). Among non-LEE-encoded effectors, NleA (also known as EspI) and NleB have been shown to be essential for the virulence of A/E pathogens (31, 46, 54, 96), while others contribute to colonization and full virulence, performing different and sometimes redundant functions within the host cell, including antiapoptotic activities, disruption of host innate immune responses by preventing NF-κB activation at different levels, increase of paracellular permeability, blockage of cell division, disruption of microtubule cytoskeleton, and inhibition of macrophage opsonophagocytosis, among others (4, 7, 13, 17, 21, 26, 34, 35, 38, 56, 57, 59, 60, 66, 69, 72, 75, 79, 84–86, 88, 94, 95, 97, 98, 100).

The expression of LEE genes is tightly regulated in A/E pathogens. Ler, encoded by the first gene of the LEE1 operon, acts as the master transcriptional regulator of the rest of the LEE operons by counteracting the repression exerted by the global regulator H-NS (2, 5, 23, 25, 32, 63, 64, 81, 87). The LEE encodes two additional regulators, GrlA, an activator of ler transcription, and GrlR, which negatively regulates the expression of LEE genes (18). Together, GrlA and GrlR appear to regulate the spatiotemporal expression of Ler and thus of all Ler-dependent promoters (2, 18, 39, 41–43, 50, 51, 55). In addition to the LEE-encoded regulators, many other factors have been implicated in the regulation of LEE-encoded genes (reviewed in references 3, 62, 73, 90, and 99).

Although the knowledge concerning the functions of non-LEE-encoded effectors is rapidly expanding, the regulatory mechanisms controlling their expression have just started to be elucidated. Recent reports indicate that the quorum-sensing regulator QseA and the two-component signaling system QseEF regulate the expression of espFu (also called tccP) in EHEC in a cascade fashion (76) and that NleA is heterogeneously expressed together with Tir and EspA in a Ler-dependent manner (77, 82). In contrast, the expression of nleG (also called nleI) in EPEC, as well as that of the espJ-espFu (tccP) operon in EHEC, has been shown not to depend on Ler (28, 54). In addition, expression of nleA, but not nleD or espJ, responds to SOS signaling in EPEC (65). We have previously demonstrated that Ler and GrlA do not regulate nleH expression at the transcriptional level in C. rodentium; however, Lon regulates its cytoplasmic levels depending on the activity of the T3SS (27). To date, no common regulatory mechanism has been described for the expression of effectors encoded outside the LEE. In this work, we describe the identification and characterization of an upstream DNA sequence motif conserved among a subset of genes encoding effector proteins in A/E pathogens, which includes an inverted repeat sequence here named NRIR (nle regulatory inverted repeat). Here, we assessed the role of the NRIR in the regulation of nleH1 and nleB2, studied the regulation of nleA, an effector gene lacking the NRIR, and evaluated the role of diverse LEE-encoded and global regulators in their transcription.

MATERIALS AND METHODS

Bacterial strains and culture conditions.

Bacterial strains and plasmids used in this study are listed 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) with or without shaking (220 rpm). When required, the medium was supplemented with ampicillin (100 μg/ml), nalidixic acid (15 μg/ml), kanamycin (Kan; 30 μg/ml), or streptomycin (100 μg/ml). DMEM was also supplemented with 1% LB broth.

Table 1.

Strains and plasmids used in this study

Strain or plasmid	Descriptiona	Reference or source
EPEC strains
E2348/69	Wild-type EPEC O127:H6, Nalr	53
JPEP24	E2348/69 derivative, Δler::kan	V. H. Bustamante, unpublished
JPEP35	E2348/69 derivative, carrying an in-frame deletion of grlA	A. Huerta, unpublished
JPEP39	E2348/69 derivative, carrying an in-frame deletion of grlR	A. Huerta, unpublished
JPEP36	E2348/69 derivative, Δhns::kan	A. Vazquez, unpublished
JPEP40	E2348/69 derivative, ΔstpA::kan	A. Vazquez, unpublished
JPEP41	E2348/69 derivative, ΔuvrY::kan	This study
JPEP42	E2348/69 derivative, ΔphoP::kan	This study
JPEP43	E2348/69 derivative, ΔompR::kan	This study
JPEP44	E2348/69 derivative, Δfis::kan	A. Vazquez, unpublished
JPEP45	E2348/69 derivative, ΔhimA::kan	A. Vazquez, unpublished
JPEP46	E2348/69 derivative, ΔrpoS:kan	A. Vazquez, unpublished
JPEP47	E2348/69 derivative, Δhha::kan	A. Vazquez, unpublished
JPEP48	E2348/69 derivative, nleA::FLAG	This study
JPEP49	JPEP48 derivative, Δler	This study
JPEP50	JPEP48 derivative, ΔgrlA	This study
JPEP51	JPEP48 derivative, ΔgrlR	This study
Plasmids
pKK232-8	pBR322 derivative containing a promoterless cat gene, Apr	Pharmacia LKB Biotech
pKKnleH1-1	pKK232-8 derivative containing nleH-cat transcriptional fusion from nucleotides −720 to +49	This study
pKKnleH1-2	pKK232-8 derivative containing nleH-cat transcriptional fusion from nucleotides −256 to +49	This study
pKKnleH1-3	pKK232-8 derivative containing nleH-cat transcriptional fusion from nucleotides −97 to +49	This study
pKKnleH1-4	pKK232-8 derivative containing nleH-cat transcriptional fusion from nucleotides −256 to +49, with −10 promoter box replaced by an ApaI restriction site	This study
pKKnleH1-5	pKK232-8 derivative containing nleH-cat transcriptional fusion from nucleotides −256 to +49, with NRIR partially replaced by SalI and XhoI restriction sites	This study
pKKnleH1-6	pKK232-8 derivative containing nleH-cat transcriptional fusion from −64 to +49 nucleotide	This study
pKKnleB2-1	pKK232-8 derivative containing nleB2-cat transcriptional fusion from nucleotides −240 to +60	This study
pKKnleB2-2	pKK232-8 derivative containing nleB2-cat transcriptional fusion from nucleotides −193 to +60, with −10 box of promoter 1 replaced by an ApaI restriction site	This study
pKKnleB2-3	pKK232-8 derivative containing nleB2-cat transcriptional fusion from nucleotides −193 to +60, with −10 box of promoter 2 replaced by an ApaI restriction site	This study
pKKnleB2-4	pKK232-8 derivative containing nleB2-cat transcriptional fusion from nucleotides −193 to +60, with −10 boxes of both promoters replaced by ApaI restriction sites	This study
pKKnleB2-5	pKK232-8 derivative containing nleB2-cat transcriptional fusion from nucleotides −130 to +60, with complete NRIR	This study
pKKnleB2-6	pKK232-8 derivative containing nleB2-cat transcriptional fusion from nucleotides −240 to +60, with NRIR partially replaced by SalI and XhoI restriction sites	This study
pKKnleB2-7	pKK232-8 derivative containing nleB2-cat transcriptional fusion from nucleotides −93 to +60	This study
pKKnleA-649	pKK232-8 derivative containing nleA-cat transcriptional fusion from nucleotides −649 to +140	This study
pKKnleA-179	pKK232-8 derivative containing nleA-cat transcriptional fusion from nucleotides −179 to +140	This study
pKKnleA-149	pKK232-8 derivative containing nleA-cat transcriptional fusion from nucleotides −149 to +140	This study
pKKnleA-123	pKK232-8 derivative containing nleA-cat transcriptional fusion from nucleotides −123 to +140	This study
pKKnleA-87	pKK232-8 derivative containing nleA-cat transcriptional fusion from nucleotides −87 to +140	This study
pKKnleA-65	pKK232-8 derivative containing nleA-cat transcriptional fusion from nucleotides −65 to +140	This study
pKKnleA-56	pKK232-8 derivative containing nleA-cat transcriptional fusion from nucleotides −56 to +140	This study
pKKnleA-47	pKK232-8 derivative containing nleA-cat transcriptional fusion from nucleotides −47 to +140	This study
pKKnleA-179ApaI	pKKnleA-179 derivative, with the −10 promoter box replaced by an ApaI restriction site	This study
pSEPZ-11	pKK232-8 derivative containing LEE2-cat transcriptional fusion from nucleotides −469 to +121	5
pRE112ΔgrlREP	Suicide construct carrying an in frame deletion of EPEC grlR in pRE112	A. Huerta, unpublished
pRE112ΔgrlAEP	Suicide construct carrying an in frame deletion of EPEC grlA in pRE112	A. Huerta, unpublished
pRE112ΔlerEP	Suicide construct carrying an in frame deletion of EPEC ler in pRE112	A. Huerta, unpublished
pMPM-T3	p15A derivative low-copy-no. cloning vector, lac promoter, Tcr	61
pTEPLer1	pMPM-T3 derivative expressing ler from the lac promoter	6
pTEPGrlA1	pMPM-T3 derivative expressing grlA from the lac promoter	6
pT3GrlR	pMPM-T3 derivative expressing grlR from the lac promoter	C. Lara-Ochoa, unpublished
pT3-HNS	pMPM-T3 derivative carrying the hns gene, Tcr	5
pKD4	pANTSγ derivative containing an FRT-flanked Kan resistance gene	14

^aThe coordinates for cat transcriptional fusions are indicated with respect to the nleH1 or nleB2 translational start sites or the nleA or LEE2 transcriptional start sites.

DNA manipulations.

Recombinant DNA techniques were performed according to standard protocols (80). Restriction enzymes were obtained from Invitrogen and used according to the manufacturer's instructions. The oligonucleotides used for PCR amplification and for primer extension experiments were synthesized by the oligonucleotide synthesis facility at the Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM). PCRs were performed in 100-μl reaction mixtures containing a 1.5:1 mixture of AmpliTaq and Pfu DNA polymerases, using an Eppendorf Mastercycler or Perkin-Elmer thermocycler.

RNA isolation and primer extension analysis.

Total RNA was isolated from samples of cultures grown for 6 h in DMEM at 37°C, using an RNeasy kit (Qiagen) according to the manufacturer's instructions. The RNA concentration and quality were determined by measuring the A₂₆₀ and by gel electrophoresis. Primer extension reactions were performed as described previously (58). Briefly, oligonucleotides complementary to the 5′ regulatory region of nleB2, nleH1, or nleA were end labeled with [γ-³²P]dATP, using T4 polynucleotide kinase, and annealed with 30 μg of total RNA in 0.37 M NaCl–0.035 M Tris-HCl (pH 7.5) by heating for 3 min at 90°C and then cooling slowly to 50°C. Reverse transcription reactions were performed at 42°C for 2 h with 10 U of avian myeloblastosis virus (AMV) reverse transcriptase (Boehringer Mannheim) in AMV reverse transcriptase buffer containing 1 mM dithiothreitol, 0.3 mM each deoxynucleoside triphosphate, and 50 U of RNase inhibitor (Invitrogen). The reverse transcription products were cleaned and concentrated using a Microcon YM-30 microconcentrator (Amicon) according to the specifications of the manufacturer, denatured by heating to 95°C for 5 min in loading buffer, and resolved by electrophoresis in an 8% polyacrylamide–7 M urea–Tris-borate-EDTA sequencing gel. The gel was analyzed using a PhosphorImager scanner (Molecular Dynamics). The transcriptional start site was determined by comparison with a DNA ladder obtained by sequencing plasmid pKKnleB2-1, pKKnleH1-1, or pKKnleA-179 with the same specific primer used for the primer extension reactions described above.

Construction of cat transcriptional fusions.

Oligonucleotides were designed to PCR amplify fragments of different lengths spanning the regulatory regions of nleH1, nleB2, and nleA (see Fig. 3A and C and 5A and B) and to introduce BamHI and HindIII restriction sites at the ends. PCRs were performed using the appropriate oligonucleotides (see Table S1 in the supplemental material) and EHEC EDL933 chromosomal DNA as the template. The PCR fragments were digested with BamHI and HindIII and ligated into pKK232-8, which contains a promoterless cat gene, digested with the same enzymes.

Fig 3.

Determination of the transcriptional start sites of NRIR-containing genes. Primer extension assay on total RNA samples of EPEC strains containing nleH1-cat (A) or nleB2-cat (B) fusions. Total RNA was isolated from samples of cultures grown in shaken DMEM for 6 h at 37°C. Oligonucleotides complementary to the 5′ regulatory region of nleH1 and nleB2 were end labeled with [γ-³²P]dATP and annealed with 30 μg of total RNA from the indicated strains. The primer extension reactions were resolved by electrophoresis in an 8% polyacrylamide–7 M urea–Tris-borate-EDTA sequencing gel. The gel was analyzed using a PhosphorImager scanner (Molecular Dynamics). The transcriptional start site was determined by comparison with a DNA ladder obtained by sequencing plasmids pKKnleH1-1 and pKKnleB2-1, as indicated in Materials and Methods. The arrows indicate the transcriptional start site and the primer extension product of each gene. Lanes PE, primer extension products.

Fig 5.

nleA belongs to the Ler regulon. CAT specific activity of the nleA-cat fusion in wild-type EPEC and its null Δler, ΔgrlA, ΔgrlR, and Δhns mutants carrying either plasmid pMPM-T3 (vector control) or the corresponding complementing plasmid pTEPLer1, pTEPGrlA1, pT3GrlR, or pT3-HNS, grown in DMEM (A) or LB medium (B). Culture samples were taken at stationary phase. Activities are expressed as percentages with respect to the wild-type strain. Results shown are the average of three independent experiments done in duplicate. Wild-type EPEC and its Δler, ΔgrlA, and ΔgrlR mutants carrying nleA chromosomally tagged with a three copies of the FLAG epitope and the corresponding complemented strains were grown in DMEM (C) or LB medium (D), and total extracts were subjected to Western blot analysis using anti-FLAG or anti-EspA antibodies. DnaK was detected as a loading control using monoclonal anti-DnaK antibodies.

To construct pKKnleB2-1, pKKnleB2-5, and pKKnleB2-7, the regulatory region of nleB2 was amplified by PCR using primer nleB2L plus either nleB2U, nleB2IRU, or nleB2DIRU, respectively, and the resulting products were cloned into the HindIII and BamHI sites of pKK232-8. Mutations in the regulatory region of nleH1 and nleB2 were introduced by the method described by Ito and collaborators (40). To generate pKKnleB2-2 and pKKnleB2-3, amplicons containing the nleB2 putative −10 P1 or P2 promoter sequence replaced by the ApaI restriction site were obtained using oligonucleotides nleB2U plus nleB2-10U and nleB2-10L plus nleB2L for P1 or nleB2U plus mutnleB2U and mutnleB2L plus nleB2L for P2 (see Table S1 in the supplemental material), respectively. Individual products were amplified, purified, mixed, and used as templates for a second amplification with external oligonucleotides (nleB2U and nleB2L). The final products were cloned into the HindIII and BamHI sites of pKK232-8. To generate the P1 and P2 double mutant where both −10 promoter sequences were replaced by an ApaI restriction site, the pKKnleB2-3 fusion was used as a template to generate complementary amplicons by PCR with oligonucleotides nleB2U/nleB2-10U and nleB2-10L/nleB2L. Individual amplicons were purified, mixed, and used as a template for a second PCR amplification with external oligonucleotides nleB2U and nleB2L. The resulting PCR fragment was cloned into the HindIII and BamHI sites of pKK232-8, yielding pKKnleB2-4. Mutations in the NRIR sequence were introduced using oligonucleotides nleB2U/nleB2IRmU and nleB2IRmL/nleB2L to amplify complementing amplicons further used to generate pKKnleB2-6, as described above.

To construct pKKnleH1-1, pKKnleH1-2, pKKnleH1-3, and pKKnleH1-6, the regulatory region of nleH1 was amplified by PCR using the oligonucleotide nleH1L containing a HindIII site plus nleH1U, nleH1U2, nleH1U3, or nleH1U6, respectively, each containing a BamHI site (see Table S1 in the supplemental material). The resulting products were cloned into the BamHI and HindIII sites of pKK232-8. To generate pKKnleH1-4, where an ApaI site replace the nleH1 putative −10 promoter sequence, two PCRs were performed using oligonucleotide pairs nleH1U/nleH1-10L and nleH1-10U/nleH1L. The resulting amplicons were mixed and used as a template for a second PCR amplification with external oligonucleotides nleH1U and nleH1L. The final PCR product was purified and cloned into the HindIII and BamHI sites of pKK232-8. Mutations in the nleH1 NRIR sequence were introduced using oligonucleotides nleH1U/nleB2IRmU and nleB2IRmL/nleH1L, and the resulting amplicons were further used to generate pKKnleH1-5, as described above.

To construct pKKnleA-649, pKKnleA-179, pKKnleA-149, pKKnleA-123, pKKnleA-87, pKKnleA-65, pKKnleA-56, and pKKnleA-47, the regulatory region of nleA was amplified by PCR using oligonucleotide nleAL containing an HindIII site plus nleAU, nleAU2, nleAU3, nleAU4, nleAU5, nleAU6, nleAU7, or nleAU8, respectively, each containing a BamHI site (see Table S1 in the supplemental material). The resulting products were cloned into the HindIII and BamHI restriction sites of pKK232-8. To generate pKKnleA-179ApaI, pKKnleA-179 was used as a template for inverse PCR using oligonucleotides nleA-10U and nleA-10L. The resulting amplicon was digested with ApaI and self-ligated.

Construction of deletion mutants and nleA FLAG-tagged strains.

Nonpolar gene deletion mutants of EPEC E2348/69 in the phoP, ompR, and uvrY genes were generated by a method using PCR-based one-step inactivation of chromosomal genes (14). PCRs to obtain mutagenic products were carried out with plasmid pKD4 as the template and primers ompRH1P1/ompRH2P2 for the ompR mutant, phoPH1P1/phoPH2P2 for the phoP mutant, and uvrYH1P1/uvrYH2P2 for the uvrY mutant. Candidates were selected in Kan, and the elimination of the genes was corroborated by PCR using oligonucleotides ompRA/ompRB for the ompR mutant, phoPA/phoPB for the phoP mutant, and uvrYA/uvrYB for the uvrY mutant. The nleA gene was FLAG tagged in EPEC, as previously described (92), using oligonucleotides nleA-FLAGH1P1 and nleA-FLAGH2P2, yielding the EPEC nleA::3XFLAG-kan strain. In-frame deletions in grlR, grlA, and ler were generated by the sacB gene-based allelic exchange method as described previously (22), using suicide clones carrying in-frame deletions of grlR, grlA, and ler (Table 1). Single and double mutants were verified by PCR and complementation analysis.

CAT assay.

Chloramphenicol acetyltransferase (CAT) specific activity from total cell extracts was determined as previously described (58).

Western blot assays.

Protein extracts from different strains were loaded onto SDS-PAGE gels and transferred to nitrocellulose membranes (0.45-μm pore size; Millipore). Membranes containing transferred proteins were blocked overnight in phosphate-buffered saline (PBS)-Tween 20 (PBST) and 5% nonfat milk at 4°C. Membranes were washed three times with PBST buffer and incubated with anti-FLAG (1:3,000) and anti-DnaK (1:10,000) monoclonal antibodies or anti-EspA (1:5,000) polyclonal antibody for 4 h with shaking at room temperature. Membranes were washed three times with PBST buffer and incubated for 1 h with rabbit anti-mouse peroxidase-conjugated or goat anti-rabbit peroxidase-conjugated (1:10,000) antibody, respectively. Positive signals were visualized with Western Lightning Chemiluminescence Reagent Plus (Perkin-Elmer). Membranes were exposed to Kodak X-Omat LS films.

Statistical analysis.

One-way analysis of variance was performed using SPSS software. Statistical differences among media were detected using Fisher's least significant difference posthoc test.

RESULTS

A subset of nle genes shares a conserved DNA motif at their regulatory regions.

In contrast to C. rodentium, EPEC and EHEC possess two functional nleH genes (27, 38). We have previously reported that the expression of the single nleH gene in C. rodentium is not Ler dependent (27). To get insights into the regulatory elements involved in nleH transcriptional regulation, the 5′ upstream region of EPEC nleH genes (nleH1 and nleH2) was aligned in search of conserved motifs. This analysis revealed the presence of a conserved 13-mer inverted repeat sequence with a TCCGG spacer upstream of the −35 hexamer of their putative promoters (Fig. 1). A BLAST search using this motif, here called the nle regulatory inverted repeat (NRIR), showed that it is only found in the genomes of A/E pathogens. As in the case of nleH2 and the nleH1-nleF putative operon, this sequence was also present mostly upstream of genes coding for other Nle proteins in A/E pathogens flanked by additional upstream and downstream sequences that were also highly conserved (Fig. 1 and Table 2). In EPEC, the NRIR was also found upstream of the nleG (nleI) gene and of the nleB2-nleC putative operon, as well as of gene E2348C_1441 of unknown function, which is located in the vicinity of nleA (E2348C_1442) and nleH1 (E2348C_1444). In addition, the NRIR, without the conserved flanking sequences, was also found downstream of a gene coding for a small predicted protein (E2348C_1263) and of the mdh malate dehydrogenase gene (E2348C_3507); however, the NRIR does not seem to have a regulatory function at this position, suggesting that it remained there as a consequence of gene rearrangements during the evolution of A/E E. coli. The NRIR is also conserved at the 5′ upstream sequence of several T3SE genes of EHEC strains EDL933 and Sakai and of C. rodentium ICC168 (Table 2), as well as of other A/E E. coli strains whose genome sequences are available (data not shown). In EHEC EDL933, a total of 10 regulatory regions controlling the expression of 14 T3SE genes (nleH2, nleH1-nleF, nleB2-nleC, a member of the espX family and 8 out of the 13 copies of nleG, as well as a truncated nleG gene [Z2076]) contain the NRIR, while the espFu (tccP) and tccP2 genes have the NRIR downstream of their coding sequences. Most genes containing the NRIR in EDL933 were also found in the Sakai strain (Table 2). Similar to the EPEC E2348/69 strain, both EHEC strains have an NRIR-like element downstream of the mdh gene and, in the case of EDL933, also at the end of dinI. Z1485 and ECs1230 are truncated orthologues of EPEC E2348C_1441, located near T3SE genes that conserve an upstream copy of the NRIR (data not shown) and therefore are likely to encode effector proteins yet to be characterized. In C. rodentium, three copies of the NRIR element are found, upstream of genes nleH-nleF, espX7, and a pseudogene copy of nleG2. The presence of the NRIR motif upstream of genes coding for different effector proteins strongly suggests the existence of a common regulatory mechanism coordinating the expression of this subset of effectors encoded outside the LEE.

Fig 1.

EPEC E2348/69 nle genes containing the NRIR. Sequence alignment of the 5′ upstream regions of nleG (nleI), nleH1, nleH2, and nleB2 genes. The 3′ end of the sequences corresponds to the putative start codon. The conserved sequence (indicated by a black line) includes the conserved 31-bp nle regulatory inverted repeat (NRIR) (dashed line). In some cases as for nleG (nleI) and nleH2, the inverted repeat is not perfect as it can either present base deletions or insertions in one half of the palindrome. Promoters and transcriptional start sites of nleB2-nleC and nleH1-nleF are indicated by black squares and arrows, respectively.

Table 2.

Genes containing the NRIR in the genomes of A/E pathogens

Gene function	NRIR-containing gene(s) by straina
	EHEC EDL933	EHEC Sakai	EPEC E2348/69	C. rodentium DBS100
T3SE	nleH1-nleF	nleH1-nleF	nleH1-nleF	nleH1-nleF
	nleH2		nleH2
	nleB2-nleC	nleB2-nleC	nleB2-nleC
	espX	espX		espX7
	Z2075 (nleG)	ECs2229 (nleG)	nleG (nleI)
	Z3921 (nleG)	ECs3488 (nleG)
	Z2149 (nleG-nleG-nleG)	ECs2156 (nleG-nleG-nleG)
	Z2339 (nleG-nleG)	ECs1994 (nleG-nleG)
	Z6025 (nleG)	ECs1811 (nleG)**
	Z2076 (nleG)**
		ECs2227 (nleG)
				nleG2
	espFu (tccP)*	ECs1126 (espFu)*
	tccP2*	ECs2715 (espFu)*
Other function	Z4595 (mdh)*	ECs4109 (mdh)*	E2348C_3507 (mdh)*
	Z1698 (dinI)*
Unknown function	Z1485**	ECs1230**	E2348C_1441
			E2348C_1263*

^a*, NRIR localizes downstream of the open reading frame; **, truncated open reading frame. A blank space means that the corresponding ortholog either is not present in the strain or lacks the NRIR. The order of the genes is arbitrary.

Effect of LEE-encoded and global regulators in expression of nleH1 and nleB2 genes.

To study the regulation of NRIR-containing effector genes, we selected nleB2 and nleH1, both present in EPEC and EHEC and sharing identical NRIRs, to construct transcriptional fusions of their regulatory regions to the promoterless cat reporter gene in plasmid pKK232-8. pKKnleB2-1 and pKKnleH1-1 contain the putative upstream regulatory regions of these nle genes, including the DNA stretch and NRIR conserved among nleH1, nleH2, nleB2, and several nleG genes (see Fig. 4A and C). In EPEC, LEE genes encoding effectors are expressed in DMEM at 37°C and repressed in LB rich medium (5, 48). In order to identify in vitro growth conditions favoring expression of nleH1 and nleB2, we cultivated wild-type (WT) EPEC bearing plasmid pKKnleH1 or pKKnleB2 in DMEM and LB medium under shaken or static growth conditions and determined CAT specific activity from samples taken at early stationary phase. Both reporter fusions displayed the highest activity in DMEM cultures under shaken growth conditions (Fig. 2A and B). The activity of the nleH1 and nleB2 promoters was reduced during static growth with respect to shaken cultures in both media. Expression during growth in M9 minimal medium was similar to that seen in DMEM (data not shown).

Fig 4.

NRIR is essential for the activation of nleH1-cat and nleB2-cat transcriptional fusions. Schematic representation of the nleH1 (A) and nleB2 (C) upstream sequences and transcriptional fusions to the cat reporter gene. The largest fusions contain the intergenic DNA region between nleH1 and a divergent integrase gene or between nleB2 and an nleG copy and its deleted and mutated derivatives lacking different cis elements. (B) CAT specific activity of culture samples of EPEC strains containing the cat reporter fusions depicted in panel A grown under both inducing (DMEM) and repressing (LB medium) conditions to stationary phase. Results shown are the averages of two independent experiments. (C) Schematic representation of the nleB2-cat reporter fusion, containing the whole intergenic region between nleB2 and nleG in EPEC, and its deleted and mutated versions. (D) CAT specific activities of culture samples of EPEC strains containing the reporter fusions depicted in panel C. Strains were grown in DMEM or LB medium to stationary phase. Results shown are the average of three independent experiments done in duplicate.

Fig 2.

Role of LEE-encoded regulators and H-NS on the expression of NRIR-containing genes. WT EPEC derivatives carrying pKKnleH1 (A) or pKKnleB2 (B) transcriptional fusions were grown under inducing or repressing conditions in LB medium or DMEM. CAT specific activity was determined for WT EPEC and its isogenic mutants in LEE-encoded regulators or H-NS, containing the nleH1-cat (C) or nleB2-cat (D) reporter fusion, grown in DMEM or LB medium. CAT specific activity was determined from culture samples taken at stationary phase. Results shown are the average of three independent experiments done in duplicate.

As effector proteins encoded by nle genes are specific substrates of the T3SS encoded by the LEE (18, 89), we sought to investigate if the expression of NRIR-containing nle genes is coordinately regulated with LEE gene expression. The LEE encodes three transcriptional regulators. Ler, encoded by the first gene of the LEE1 operon, and GrlA, encoded by the grlRA operon, are required for the transcription of the genes encoding the structural components of the T3SS and its cognate effector proteins located in this pathogenicity island (2, 5, 18, 23, 62, 64), while GrlR acts as a transcriptional repressor of the LEE (2, 18, 51, 55). In order to determine if the transcription of NRIR-containing genes is under the control of LEE-encoded regulators, the activity of nleH1-cat and nleB2-cat transcriptional fusions, contained in plasmids pKKnleH1-1 and pKKnleB2-1, respectively, was determined in ler, grlA, and grlR mutant derivatives of EPEC E2348/69 (Table 1) grown under inducing (DMEM) and repressive (LB medium) culture conditions. A small yet statistically significant reduction in CAT specific activity was seen in the Δler and ΔgrlA mutants for the nleH1-cat fusion compared to the WT growing under inducing conditions, while no differences were found among the aforementioned strains under repressive conditions (Fig. 2C). For the nleB2-cat fusion, no differences in the ler mutant compared to the WT were found under either condition, while a small but significant increase in the ΔgrlA mutant in DMEM was seen (Fig. 2D). Although a clearer regulatory effect was detected in the EPEC ΔgrlR strain growing in DMEM for both fusions, the greatest difference in expression levels was observed in this mutant growing in LB medium. In this case, 8.5- and 7-fold increases in transcription were observed for nleH1-cat and nleB2-cat fusions, respectively, compared to WT levels. These data show that GrlR acts as a negative regulator of nleH1 and nleB2, which is consistent with its repressor role in LEE gene regulation (C. Lara-Ochoa et al., unpublished data).

We also investigated the role of the global regulator H-NS in the regulation of these genes, as this protein also negatively regulates the expression of LEE genes (5, 91), and of other genes that have been acquired by horizontal gene transfer events (70). An increase in CAT activity was observed in the hns mutant strain for both genes compared to the WT strain (Fig. 2C and D). While about a 2-fold increase in CAT activity was seen for nleH1, for nleB2 there was, depending on the growth medium, a 3- to 4-fold induction in the absence of H-NS. These results showed that transcription of nleH1 and nleB2 is repressed directly or indirectly by H-NS. We also measured the expression levels of these fusions in EPEC E2348/69 mutants with mutations in global regulators such as StpA, Fis, IHF, Hha, OmpR, PhoP, UvrY, and RpoS, which are known to be involved in the regulation of the LEE or other bacterial virulence factors (16, 20, 24, 30, 44, 62). No significant difference was observed in the expression levels of nleH1 and nleB2 in the absence of any of these regulators (data not shown).

Analysis of the regulatory sequences of nleH1 and nleB2.

As inverted repeats often act as binding sites for regulatory proteins, we studied the role of the upstream conserved region and the NRIR in the regulation of nleH1 and nleB2. First, to identify their transcriptional start sites, we carried out primer extension analysis using total RNA from strains carrying pKKnleH1-1 and pKKnleB2-1 grown in DMEM (Fig. 3). Based on the transcriptional start site determined for each gene, we predicted their putative −10 and −35 promoter hexamers (P1 for nleH1-nleF and P2 for nleB2-nleC) (Fig. 1). To assess the role of the NRIR and to identify possible cis-acting elements in the expression of nleH1 and nleB2, as well as to confirm the functionality of the promoters identified, we performed a deletion and site-directed mutagenesis analysis. pKKnleH1 and pKKnleB2 derivatives (Fig. 4A and C) were transformed into wild-type EPEC, and CAT specific activity was determined in samples collected from cultures grown under inducing (DMEM) or repressing (LB medium) conditions. Expression levels of the two larger nleH1 fusions (pKKnleH1-1 and pKKnleH1-2), containing the NRIR and additional conserved DNA sequence, were similar to those observed for pKKnleH1-3, which contains only the NRIR (Fig. 4B). In contrast, the fusion contained in pKKnleH1-6, which includes the −35 and −10 putative promoter boxes but lacks the upstream NRIR, was poorly active (Fig. 4A and B). These results suggested that the NRIR has an essential role in nleH1 activation. To further confirm the role of the NRIR and of the predicted promoter in nleH1 expression, site-directed mutants of pKKnleH1-2 were generated. In pKKnleH1-4 the putative −10 hexamer was replaced by the sequence corresponding to the ApaI restriction site, while in pKKnleH1-5 the inverted repeat was replaced with restriction sites for SalI and XhoI. Activity of the nleH1 promoter was highly reduced for both fusions (Fig. 4B). Together, these results show that the NRIR is essential for nleH1 expression and that the promoter identified by primer extension analysis drives the expression of nleH1.

The transcriptional start site of nleB2 led to the prediction of a promoter sequence further downstream of the NRIR (P2 in Fig. 1) with respect to the promoter identified for nleH1. However, the corresponding −10 and −35 hexamers of the nleH1 promoter were perfectly conserved in the upstream sequence of nleB2 following the NRIR (P1 in Fig. 1). Thus, although P1 promoter activity was undetectable by primer extension, we speculated that this promoter was also functional in nleB2. To test this hypothesis and to corroborate the functionality of the promoter inferred by primer extension analysis (P2 in Fig. 1), we replaced independently the putative −10 box of each promoter by an ApaI site in pKKnleB2-1. The resulting plasmids, pKKnleB2-2 and pKKnleB2-3 (Fig. 4C), displayed a partial reduction of CAT activity in WT EPEC grown in DMEM (Fig. 4D), suggesting that the transcriptional activity of nleB2 was cooperatively driven by both promoters. Confirming this possibility, a transcriptional fusion simultaneously mutated in the −10 elements of both promoters (pKKnleB2-4 plasmid) was almost inactive under inducing conditions. Collectively, these data demonstrated that the two nleB2 promoters are functional.

Results obtained from the analysis of the deleted versions of the nleB2 regulatory region resembled those obtained for nleH1. A deleted fusion containing only the NRIR (pKKnleB2-5) is expressed at levels similar to those of the larger fusion (pKKnleB2-1), while the disruption of the inverted repeat rendered the fusion inactive in both DMEM and LB medium (Fig. 4C and D, plasmid pKKnleB2-6). These results indicated that, as for nleH1, the NRIR motif is essential, in this case for the activation of the two putative promoters identified for nleB2. Interestingly, elimination of the NRIR and P1 promoter (pKKnleB2-7) did not completely abolish the expression of the P2 promoter, which still showed partial activation (Fig. 4D), suggesting that along with the NRIR and the P1 promoter, putative P2 negative regulatory sequences were also eliminated in this fusion.

Overall, the results described above indicate that the NRIR has an essential role in the transcriptional activation of these genes.

nleA expression in EPEC is coordinately regulated with the LEE.

Except for the NRIR-containing genes, no other group of effector genes in EPEC shares DNA sequence identity in their regulatory regions (data not show). One of these genes is nleA, encoding the effector protein NleA (EspI), which is essential for C. rodentium virulence in mice (18, 31, 68). NleA is known to localize in the Golgi apparatus (11, 31) and to interact with PDZ domain-containing host proteins (52). NleA from EPEC reduces host cellular protein secretion by directly interacting with Sec24, a subunit of the COPII protein trafficking complex required for the formation of intracellular transport vesicles (49), activity that is related to its capacity to disrupt epithelial tight junctions (88).

In EHEC it has been shown that Ler regulates nleA expression (1, 77, 82), while overexpression of LEE-positive regulators such as GrlA in C. rodentium and Pch in EHEC enhanced NleA expression and secretion (18, 89). In order to contrast the regulation of NRIR-containing genes with that of an nle gene putatively regulated by Ler, we confirmed and further characterized the role of LEE-encoded regulators in the regulation of nleA. First, we constructed a transcriptional cat reporter fusion to the upstream regulatory region of nleA in pKK232-8. The resulting plasmid (pKKnleA-649) contains the entire intergenic region between nleA and the divergently transcribed E2348C_1443 integrase gene (see Fig. 7A). This plasmid was transformed into WT EPEC, and its expression was analyzed under the same growth conditions tested for nleH1 and nleB2. The absolute expression of nleA-cat was higher in DMEM than in LB medium (data not shown). Then, the expression of this construct was determined in EPEC Δler, ΔgrlA, ΔgrlR, and Δhns mutants (Table 1), grown in DMEM or LB medium, which were used as inducing or repressing growth conditions, respectively. As a control, we determined in the same strains the expression of the LEE2-cat transcriptional fusion (pSEPZ-11) (Table 1), which carries an LEE promoter that is regulated by the LEE-encoded regulators (5). The expression of nleA was drastically reduced in the ler and grlA mutants when the strains were grown in DMEM (Fig. 5A). In contrast, under repressing conditions (LB medium), the expression of nleA-cat showed a 4-fold induction in the grlR and hns mutants. These results resembled those obtained with the LEE2-cat reporter (data not shown), whose expression was, as expected, drastically reduced in the Δler and ΔgrlA mutants under inducing conditions (5, 18) and derepressed in the ΔgrlR and Δhns mutant backgrounds under repressing conditions (51). Complementation of all mutants with plasmids carrying the corresponding wild-type genes reverted the effect of the mutations on nleA-cat expression (Fig. 5A and B).

Fig 7.

Determination of the transcriptional start site of nleA. The primer extension assay was performed as indicated for Fig. 3 and in Materials and Methods. The arrows indicate the transcriptional start site and the primer extension product (lane PE).

To further corroborate the role of the LEE-encoded regulators in nleA expression at the protein level, the sequence coding for the FLAG tag was added to the 3′ end of the chromosomal nleA gene in WT EPEC and its Δler, ΔgrlA, and ΔgrlR mutant derivatives, as described in Materials and Methods. The resulting strains (Table 1) and their complemented derivatives were grown under inducing (DMEM) and repressing (LB medium) conditions, and samples were taken to determine NleA-FLAG expression in whole-cells lysates by Western blotting. In agreement with the nleA-cat transcriptional fusion data, under inducing conditions, NleA-FLAG was poorly detected in Δler strain extracts and showed reduced amounts in the ΔgrlA mutant, while in the ΔgrlR mutant a clear increase in NleA-FLAG concentration was observed (Fig. 5C). Under repressing conditions, NleA-FLAG expression was highly increased in the ΔgrlR mutant, while the rest of the strains including the wild-type showed barely detectable levels of NleA (Fig. 5D). These regulatory phenotypes were reverted in the complemented strains. Importantly, the pattern of expression of NleA resembled that of the LEE-encoded translocator EspA (Fig. 5C and D). Taken together, these results further demonstrate that nleA expression is regulated by the LEE-encoded regulators as well as by the global regulator H-NS and therefore coregulated with LEE gene expression.

Identification of nleA cis-acting regulatory elements.

To get further insights into the regulation of nleA, we performed a deletion analysis by generating nleA-cat fusions encompassing different lengths of its 5′ upstream regulatory region. The resulting nleA-cat fusions were named pKKnleA-649, -179, -149, -123, -87, -65, -56, and -47 (Fig. 6A and Table 1) according to their 5′ ends with respect to the nleA transcriptional start site (see Fig. 7). All fusions contained a common 3′ end corresponding to position +140 (Fig. 6A; see also Fig. 7B). These plasmids were introduced into wild-type EPEC, and the resulting strains were grown under inducing (DMEM) and repressing (LB medium) conditions. CAT specific activity was determined from samples taken at stationary phase. Fusions pKKnleA-649 and pKKnleA-149 were expressed at similar levels under both growth conditions (Fig. 6B). However, as the nleA regulatory region was further reduced, the negative regulation exerted by growth in LB medium was no longer observed (Fig. 6B). The reduction in activity observed for pKKnleA-123 under inducing conditions was statistically significant compared with that of pKKnleA-149 and suggested that the sequence located between positions −149 and −123 contains a cis-acting element required for the positive regulation of nleA. Furthermore, the transcriptional activities of the fusions contained in pKKnleA-87, pKKnleA-65, and pKKnleA-56 were similar to activity of pKKnleA-149 under inducing growth conditions (DMEM) and even slightly higher in LB medium than in DMEM (Fig. 6B). Interestingly, the promoter activity shown by these three fusions was about 3-fold greater than that seen for pKKnleA-149 and pKKnleA-123 under repressing conditions, indicating that a negative cis-acting regulatory element is located between positions −123 and −87 (Fig. 6A). A further deletion down to position −47 (pKKnleA-47) abolished nleA promoter activity in both DMEM and LB medium, indicating the presence of a cis-acting sequence between positions −56 and −47 that is required for activation of nleA.

Fig 6.

Deletion analysis of the nleA regulatory region. (A) Schematic representation of the E2348C_1443-nleA intergenic region in EPEC E2348/69 and of the nleA-cat transcriptional fusions generated in this work. The nleA-cat fusions were named according to the position of the 5′ end of the nleA region contained in each fusion with respect to the transcriptional start site. Positive and negative regulatory elements are indicated. (B) CAT specific activity of culture samples of EPEC containing the reporter nleA-cat fusions grown under both inducing (DMEM) and repressing (LB medium) conditions for 10 h. (C) nleA-cat reporter fusions were introduced in E. coli K-12 strain MC4100 bearing plasmid pMPM-T3 or pTEPLer1, encoding EPEC Ler. Strains were grown in DMEM at 37°C, and CAT specific activity from culture samples taken at stationary phase was determined. (D) WT EPEC or its Δler mutant derivative carrying selected nleA-cat reporter fusions was grown in DMEM at 37°C. CAT specific activity from samples taken at stationary phase was determined. Results shown are the average of three independent experiments. Bars with statistically significant differences (P < 0.05) compared with the larger fusion in the same strain or under the same growth condition are marked with an asterisk. In addition, other relevant significant P values among bars are specifically indicated.

To further characterize the role of Ler and the cis-acting elements controlling the expression of nleA, we analyzed the expression of the nleA-cat fusions in E. coli K-12, which naturally lacks ler, in the presence of either pMPM-T3 (empty vector) or pTEPLer1 carrying full-length EPEC ler (Table 1). Fusions pKKnleA-649, pKKnleA-179, and pKKnleA-149 were expressed at very low levels in the presence of the empty vector but were significantly activated in a Ler-dependent manner (Fig. 6C). In contrast, fusion pKKnleA-123 was no longer responsive to Ler, suggesting that Ler requires the sequence upstream of position −123 to activate nleA expression. Furthermore, CAT expression was restored for fusions pKKnleA-87, pKKnleA-65, and pKKnleA-56 in a Ler-independent manner (Fig. 6C), confirming the role of the sequence located between positions −123 and −85 in the negative control of nleA. The results described above were further confirmed by analyzing the expression levels of a subset of the nleA-cat fusions in an EPEC Δler strain. In agreement with the results obtained in E. coli K-12, fusions pKKnleA-649 and pKKnleA-149 showed low levels of expression in this strain, while expression of pKKnleA-123 was not responsive to Ler. nleA promoter activity was increased 2- to 3-fold for fusions pKKnleA-87 and pKKnleA-65 with respect to pKKnleA-123 (Fig. 6D). These results were consistent with the role of Ler as a positive regulator of nleA expression and of the sequence upstream of position −123 in the Ler-mediated activation of this promoter. Furthermore, the increase in nleA promoter expression seen for fusions lacking sequence upstream of position −87 was also consistent with the notion that the region between positions −123 and −87 includes a negative cis-acting regulatory element (Fig. 6D).

The transcriptional start site of nleA was identified by primer extension analysis using total RNA obtained from wild-type EPEC containing pKKnleA-179 (Fig. 7A) grown in DMEM. A transcriptional start site was identified 80 bp upstream of the nleA start codon, which allowed the prediction of the putative −35 and −10 promoter hexamers (Fig. 7B). To further confirm the functionality of this promoter, the putative −10 hexamer was replaced by the ApaI restriction site by site-directed mutagenesis in pKKnleA-179. The resulting fusion, pKKnleA-179ApaI (Fig. 6A), was inactive (Fig. 6B), confirming the functionality of the predicted nleA promoter.

Ler and H-NS interact with the regulatory region of nleA.

Ler acts as an antagonist of H-NS by directly interacting with DNA in the vicinity of the region where H-NS exerts its negative effect (reviewed in references 62 and 90). To determine if Ler and H-NS regulate nleA transcription by directly binding to its regulatory region, purified Ler-Myc-His₆ and H-NS–FLAG-His₆ recombinant proteins were used to perform electrophoretic mobility shift assays (EMSA) with PCR fragments spanning different lengths of the nleA regulatory region (Fig. 8A). As shown in Fig. 8B, Ler-Myc-His₆ interacted with the −649 to +140 and the −149 to +140 fragments in a concentration-dependent manner. Consistent with the gradual decreased in the Ler-dependent activation observed with fusions pKKnleA-649, pKKnleA-179, and pKKnleA-149 (Fig. 6B to D), Ler bound to the −649 to +140 fragment with higher affinity than to the −149 to +140 fragment, suggesting that Ler, upon binding to its putative nucleation binding site, may extend further upstream by oligomerization. H-NS–FLAG-His₆ interacted with the same fragments as Ler, suggesting that these proteins bind to DNA motifs that might be in close proximity (Fig. 8C); however, according to the deletion analysis, the H-NS nucleation binding site is located slightly downstream from the Ler binding site as a fusion carrying sequence up to position −123, while still partially repressed by H-NS, was no longer responsive to Ler (Fig. 6). No interaction of either Ler or H-NS was observed with fragments containing sequence up to position −87 or −56 (Fig. 8), which is consistent with the constitutive and unregulated expression of the nleA promoter observed with or without Ler (Fig. 6).

Fig 8.

Ler and H-NS interact with the regulatory region of nleA. (A) Schematic representation of the nleA upstream region and of the fragments used in EMSA. (B and C) EMSA of the indicated DNA fragments spanning different lengths of the nleA regulatory sequence. DNA fragments were mixed with increasing concentrations of purified Ler-Myc-His₆ (B) or H-NS-FLAG-HIS₆ (C). Free DNA and protein-DNA complexes were resolved by 5% polyacrylamide gel electrophoresis and stained with ethidium bromide.

DISCUSSION

The genomes of A/E pathogens possess a variable number, ranging between 21 and 50, of functional genes coding for type III effector proteins. Most of these genes are located outside the LEE pathogenicity island within lambda-like prophages or integrative elements and are generically called non-LEE-encoded effectors or Nles (18, 38, 74, 89). Although not all A/E effectors have been functionally characterized, it is now evident that these proteins exert diverse, cooperative, and redundant functions once translocated into the host cells and have variable degrees of importance during infection (15). To coordinate the spatiotemporal expression of these T3SE genes during the course of an infection, regulatory mechanisms would have been acquired to allow their coexpression with other LEE genes in order to have the effectors readily available for secretion upon assembly of the T3SS. The expression of LEE genes is regulated by a complex assortment of global regulators but is mainly under the positive control of the LEE-encoded regulators Ler and GrlA (reviewed in references 62, 90, and 99).

In this work, we identified the presence of a common novel DNA motif (here, denominated NRIR) located in the regulatory regions of several nle genes coding for different effector proteins of A/E pathogens. This conserved DNA region consists of a 13-bp inverted repeat separated by a conserved 5-bp spacer. NRIR-containing genes include nleG (nleI; also other nleG paralogs), nleH1, nleH2, and nleB2. It is worth noting that although the DNA stretch conserved among nle genes extends beyond the NRIR, reporter fusions carrying only the inverted repeats were expressed at the same levels as the largest fusions. Moreover, elimination of the NRIR by deleting it or by replacing 6 bp within the inverted repeats completely abolished the activity of NRIR-containing promoters, indicating that this is an essential element of the regulatory regions of this subset of nle genes. It remains to be determined if the conserved sequence upstream of the NRIR has a regulatory role under growth conditions not tested in this work. Dimeric regulatory proteins often bind to DNA inverted repeat upstream promoters to control gene transcription. In this context, the essential role of the NRIR in the transcription of NRIR-containing genes suggests that a regulatory protein, not yet identified, may target this sequence to positively regulate and coordinate the expression of these genes.

A/E pathogens acquired and developed a fine-tuned regulatory network to control the appropriate spatiotemporal expression of LEE-encoded functions such as the T3SS. Upon the discovery of non-LEE-encoded effectors, it was hypothesized that their expression might be coregulated with the T3SS (65, 77); however, as shown here, Ler does not regulate the transcription of nleB2, and a minor effect was seen on the expression of nleH1 in EPEC, in agreement with our previous studies indicating that the nleH gene in C. rodentium, which also contains a copy of the NRIR, is transcribed mainly in a Ler- and GrlA-independent manner (27). More recently, GrlA and Ler were shown to regulate the expression of both nleH1 and nleH2 in EHEC, albeit to different extents with respect to each other and to tir (36). However, it remains to be analyzed in further detail whether the nleH genes can be differentially regulated by Ler depending on the A/E pathotype or the growth conditions since growth of EHEC in DMEM seems to diminish the expression of the nleH genes (36), while for EPEC this medium was found to favor their expression (this work). Furthermore, in EPEC, the expression of nleG (nleI), which has a copy of the NRIR upstream of its putative promoter, was also previously shown to be independent of Ler (54), as well as the expression of the EHEC effector genes espJ and espFu (tccP) (28). In contrast, espFu (tccP) is regulated by the QseEF two-component system (76) and by Hfq, which also affects the expression of several other nle genes (83).

Along these lines, the observed putative repression exerted by GrlR and H-NS on the studied nle genes (Fig. 2) (27) may allow their coordinate expression with the LEE genes, which are also repressed by GrlR (51) and H-NS (2, 5, 32, 50, 81, 91). Although nleH1 and nleB2 expression in EPEC is not Ler dependent, their repression by H-NS is consistent with the proposal that this global regulator predominantly represses genes contained in horizontally acquired DNA (71), such as that of prophages, where most NRIR-containing genes are encoded (38, 74, 89).

The key role that NleA plays in the virulence of A/E pathogens is consistent with the high degree of association observed between the nleA gene and the LEE in Shiga toxin-producing E. coli (STEC) and EPEC strains of different serotypes and origins (9, 10, 12, 68). Practically all nleA genes analyzed to date are phage encoded, and the comparison of their nucleotide sequences has shown that there are at least 15 allelic variants and that the gene content of the nleA flanking regions can be classified into four groups based, for example, on the presence of other nle genes (10, 12). Despite this variability, the regulation of nleA by Ler seems to be a conserved feature since it is at least the case for variants nleA of STEC strain 4795/97 (O84:H4) (nleA₄₇₉₅) (82), nleA8-2 of EPEC E2348/69 (O127:H6) (this work), and Z6024 of EHEC NCTC12900 (O157:H7) (77); however, we cannot rule out the possibility that regulatory variations may be found among orthologs of different strains, as has been recently illustrated for the role that Hfq plays in the regulation of the LEE (47) or for the regulation of a number of nle genes in EHEC, including nleH1 and nleB2 that seem to be targets of Pch regulators, which are not present in EPEC or C. rodentium (1).

Here, we showed that Ler and H-NS exert their regulatory effects by directly binding to the regulatory sequence of nleA. Ler and H-NS binding sites seem to overlap around position −123 with respect to the identified transcriptional start site in a fashion that resembles the interaction of these two proteins at other Ler-regulated promoters (2, 5, 32, 78, 81, 91). In this manner, expression of nleA will be induced mostly under conditions that allow the expression of Ler and, thus, of the LEE genes, where it overcomes H-NS repression by competitively binding to a region that overlaps the H-NS binding sites at the LEE promoters. Consistent with a recent report (82), GrlA was also required for full activation of nleA, most likely in an indirect manner as GrlA's role in the regulation of LEE genes is due to its function as a positive regulator of ler expression (2, 18, 37, 39, 42, 50).

This study compares two of the mechanisms that regulate the expression of nle genes in EPEC. In contrast to the Ler-dependent regulation of nleA expression, other nle genes such as nleH1 and nleB2 are independently controlled by a mechanism involving the conserved NRIR motif although other strategies were developed for other nle genes during the evolution of A/E pathogens, as illustrated by the espJ-espFu (tccP) operon in EHEC, which is regulated by the LysR-type regulator QseA, and the two-component signaling system QseEF in a cascade fashion (28, 76). Altogether, these observations illustrate that a variety of mechanisms was adopted by A/E pathogens to control the expression of these genes and that this diversity perhaps reflects acquisition at different evolutionary times or the need to coregulate their expression in response to a particular environmental signal or during a specific stage of the infection. However, it is interesting that both nleH1 and nleB2 are better induced in DMEM cultures but repressed in LB rich medium, like the LEE genes and nleA, suggesting that even though they are controlled by different regulatory elements, their expression may be coregulated by convergent environmental signals that ensure their coexpression with, for example, those genes required for the assembly and function of the T3SS.