Atypical Enteropathogenic Escherichia coli That Contains Functional Locus of Enterocyte Effacement Genes Can Be Attaching-and-Effacing Negative in Cultured Epithelial Cells

Abstract

Enteropathogenic Escherichia coli (EPEC) induces a characteristic histopathology on enterocytes known as the attaching-and-effacing (A/E) lesion, which is triggered by proteins encoded by the locus of enterocyte effacement (LEE). EPEC is currently classified as typical EPEC (tEPEC) and atypical EPEC (aEPEC), based on the presence or absence of the EPEC adherence factor plasmid, respectively. Here we analyzed the LEE regions of three aEPEC strains displaying the localized adherence-like (LAL), aggregative adherence (AA), and diffuse adherence (DA) patterns on HEp-2 cells as well as one nonadherent (NA) strain. The adherence characteristics and the ability to induce A/E lesions were investigated with HeLa, Caco-2, T84, and HT29 cells. The adherence patterns and fluorescent actin staining (FAS) assay results were reproducible with all cell lines. The LEE region was structurally intact and functional in all strains regardless of their inability to cause A/E lesions. An EspF_U-expressing plasmid (pKC471) was introduced into all strains, demonstrating no influence of this protein on either the adherence patterns or the capacity to cause A/E of the adherent strains. However, the NA strain harboring pKC471 expressed the LAL pattern and was able to induce A/E lesions on HeLa cells. Our data indicate that FAS-negative aEPEC strains are potentially able to induce A/E in vivo, emphasizing the concern about this test for the determination of aEPEC virulence. Also, the presence of EspF_U was sufficient to provide an adherent phenotype for a nonadherent aEPEC strain via the direct or indirect activation of the LEE4 and LEE5 operons.

INTRODUCTION

Diarrheal diseases are responsible for about 2 million deaths of children all over the world per year, mainly in developing countries (7). Enteropathogenic Escherichia coli (EPEC) is among the leading agents of acute infantile diarrhea (25, 28, 57). EPEC has been classified into two subgroups, named typical EPEC (tEPEC) and atypical EPEC (aEPEC), based on the presence or absence of the EPEC adherence factor plasmid (pEAF), respectively (27).

The hallmark of EPEC pathogenesis is the ability to cause the attaching-and-effacing (A/E) lesion, which results from intimate bacterial adhesion to the intestinal epithelium, the effacement of local microvilli, and the accumulation of polymerized actin and other cytoskeleton elements at the site of bacterial attachment, forming pedestal-like structures (42). The capacity to cause A/E in vitro can be verified by the fluorescent actin staining (FAS) test, which detects polymerized actin aggregation at the site of bacterial attachment (33).

A/E lesion-related genes are located in a pathogenicity island named the locus of enterocyte effacement (LEE) (28, 38). The LEE is organized into 5 operons (LEE1 to LEE5), where LEE1 to LEE3 encode type III secretion system (T3SS) proteins and the LEE-encoded regulator (Ler) (18, 40), LEE4 encodes the secreted proteins that form the external part of the T3SS used to translocate effector proteins to the host cell, and LEE5 encodes the adhesin intimin and its translocated receptor Tir (reviewed in reference 20). Finally, bacteria intimately adhere to the host cell by Tir-intimin interactions, causing a cytoskeletal rearrangement that result in pedestal-like structures (30). After insertion into the eukaryotic membrane, Tir of the prototype tEPEC strain (E2348/69) is phosphorylated at tyrosine 474 at the C-terminal domain. After phosphorylation, the mammalian protein Nck is recruited to the site of bacterial adherence, where it activates the neural Wiskott-Aldrich syndrome protein (N/WASP), leading to Arp2/3 complex-mediated actin polymerization (8). In enterohemorrhagic E. coli (EHEC), another A/E-forming pathogen, Tir is not phosphorylated after the interaction with intimin (14). Instead, the bacterial effector EspF_U or TccP (Tir cytoskeleton-coupling protein) mimics the Nck protein (9, 21), triggering the recruitment of α-actin and its consequent polymerization. EspF_U indirectly couples with Tir, and IRSp53 and/or IRTKS links these two proteins (59, 62). After this interaction EspF_U activates Arp2/3 via direct and/or indirect N/WASP for actin pedestal formation (60). EPEC can induce A/E lesions using the Nck and/or EspF_U pathway or via Nck/EspF_U-independent mechanisms (3, 19).

aEPEC adherence to epithelial cells in culture is often observed as the localized adherence-like (LAL) pattern, where the adherent bacteria form loose clusters on the cell surface (1, 17, 24, 48, 50, 51, 53, 58). Some aEPEC strains may express the localized adherence (LA) pattern in 6-h assays, displaying compact clusters on the cell surface (1, 24, 26, 58); the aggregative adherence (AA) pattern, where the bacteria adhere in a stacked-brick pattern, forming aggregates on the cell surface and on the coverslip (1, 17, 24, 50, 58); and the diffuse adherence (DA) pattern, where the bacteria adhere diffusely to the cell surface (1, 17, 24, 58). Nonadherent (NA) aEPEC strains have also been reported (1, 17, 24, 50, 52).

The current study analyzed the structures and functions of the LEE regions of four aEPEC strains previously classified as being FAS positive (LAL and DA strains) and FAS negative (AA and NA strains) in HEp-2 cells. Adherence patterns and the capacity to induce A/E were examined by using different human epithelial cell lines and showed that the origin of the cell lines had no influence on both phenotypes. As LEE was present and functional in all strains, we investigated the influence of EspF_U on espF_U-negative strains, demonstrating its indirect role in bacterial adhesion to epithelial cells in vitro.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

E. coli strains and plasmids used in the present study are listed in Table 1. The four aEPEC strains were isolated from cases of acute diarrhea and previously characterized by HEp-2 cell adherence assays as displaying the LAL (strain BA320), AA (strain Ec292/84), and DA (strain 9100/83) patterns and as being unable to adhere (strain BA4013) (1, 5, 29). Also, by means of the fluorescent actin staining (FAS) test, the strains were previously classified as being FAS positive (strains BA320 and 9100/83) and FAS negative (strains Ec292/84 and BA4013) (1, 5, 29). Bacterial strains were aerobically grown in Luria-Bertani (LB) broth or Dulbecco's modified Eagle medium (DMEM) at 37°C for 18 h unless otherwise stated. Kanamycin was used at a concentration of 50 μg/ml when necessary. All strains were kept in LB broth supplemented with 15% glycerol at −80°C.

Table 1.

Bacterial strains and plasmids used in this study

E. coli strain or plasmid	Serotype	Relevant descriptionb	Reference or source
Strains
E2348/69	O127:H6	EPEC prototype strain	36
DH5α	E. coli K-12 strain	Stratagene
CVD206	O127:H6	E2348/69 eae nonpolar mutant	15
EPEC Δtir	O127:H6	E2348/69 tir nonpolar mutant	31
UMD872	O127:H6	E2348/69 espA nonpolar mutant	32
UMD864	O127:H6	E2348/69 espB nonpolar mutant	16
UMD870	O127:H6	E2348/69 espD nonpolar mutant	34
Ec292/84	O125ac:H6	aEPEC WT strain (AA)a	5
9100/83	O55:H7	aEPEC WT strain (DA)a	51
BA320	O55:H7	aEPEC WT strain (LAL)a	1
BA4013	O88:HNM	aEPEC WT strain (NA)a	1
Ec292/84(pKC471)	O125ac:H6	WT strain + pKC471	This study
9100/83(pKC471)	O55:H7	WT strain + pKC471	This study
BA320(pKC471)	O55:H7	WT strain + pKC471	This study
BA4013(pKC471)	O88:HNM	WT strain + pKC471	This study
Plasmid
pKC471		espFU-myc in pK187	9

^aAdherence pattern presented in 6-h adherence assays with HeLa cells.

^bAA, aggregative adherence; DA, diffuse adherence; LAL, localized adherence-like; NA, nonadherent.

Epithelial cell adherence and FAS assays.

The following epithelial cell lines were used: HeLa (human cervix adenocarcinoma), HEp-2 (human larynx carcinoma), Caco-2 (human colorectal adenocarcinoma), T84 (human colorectal adenocarcinoma), and HT29 (human colon adenocarcinoma) cells. HEp-2 and HeLa cells were cultivated in 24-well plates containing coverslips in DMEM supplemented with 10% fetal bovine serum (FBS) for 48 h (60 to 70% confluence). Caco-2, T84, and HT29 cells were cultivated in minimal essential medium (MEM), MEM-F12 medium, and DMEM containing 5 mM galactose, respectively, and supplemented with 10% FBS for 10 to 12 days (polarized cells) in a 10% O₂-90% CO₂ atmosphere at 37°C.

Adherence assays using different cell lines were performed for 6 h at 37°C, according to a method originally described by Cravioto et al. (11) for HEp-2 cells. Cells cultivated for 48 h in 24-well plates containing coverslips were infected with bacterial cultures statically grown overnight in LB broth (37°C), diluted 1:50 in a final volume of 1 ml/well of MEM supplemented with 2% FBS and 1% d-mannose. After 3 h of incubation at 37°C, preparations were washed with phosphate-buffered saline (PBS), and fresh medium was added. After another 3 h of incubation, preparations were washed with PBS, fixed with methanol, stained with May-Grümwald/Giemsa stain, and examined by light microscopy. The adherence patterns were classified as LAL, AA, DA, or NA according to characteristics observed and described elsewhere previously (45, 51, 54).

The ability of the strain to aggregate actin in vitro was searched by means of the FAS test as described previously by Knutton et al. (33). After the adherence assay was performed as described above, the preparations were fixed in 4% formalin, washed with PBS, treated with 0.1% Triton X-100, and washed again with PBS. Preparations were then incubated with 5 μg/ml of fluorescein isothiocyanate (FITC)-labeled phalloidin (Sigma) for 45 min. After being washed and mounted onto slides, preparations were visualized and photographed with an epifluorescence microscope (Axioskop; Carl Zeiss) connected to a digital camera (DFC300 FX; Leica). E. coli E2348/69 and DH5α were used as positive and negative controls, respectively.

Tyrosine phosphorylation of Tir.

The immunofluorescence (IFL) assay was performed according to a protocol described previously by Hernandes et al. (26). After the adherence assays were performed, preparations were fixed with 4% formalin, permeabilized with 0.1% Triton X-100, and treated with monoclonal anti-phosphotyrosine clone PT66 antibody (Sigma-Aldrich) at a dilution of 1:50, followed by goat anti-mouse IgG conjugated with FITC (Sigma-Aldrich) at a dilution of 1:25. After several washings with PBS, preparations were mounted onto slides and examined with an epifluorescence microscope (Axioskop; Carl Zeiss) connected to a digital camera (DFC300 FX; Leica).

Detection of EPEC genes.

PCRs were used to detect 34 genes of the LEE, tccP, and tccP2 (see Table S1 in the supplemental material). The PCR mixture consisted of a 25-pmol sample of each primer added with the following reagents (Invitrogen): Taq DNA polymerase (1.5 U); 10× PCR buffer (200 mM Tris-HCl [pH 8.4], 500 mM KCl) (5 μl); dATP, dCTP, dGTP, and dTTP (0.1 mM each); and MgCl₂ (2 mM). Primers, amplification cycles, sizes of amplified fragments, and control strains are also described in Table S1 in the supplemental material. Slot blot assays were used in order to verify the presence of the LEE genes not detected by PCR. Genomic DNAs of these strains, extracted as previously described (4), were hybridized with DNA probes corresponding to the genes espH, sepQ, escJ, cesF, espB, escF, grlA, cesD2, espG, espD, and espF. The probes were obtained by PCR amplification, as described above, using the genomic DNA of tEPEC prototype strain E2348/69 as a template. The amplified products were labeled by using the ECL nucleic acid labeling and detection system (GE Healthcare) according to the instructions provided by the manufacturer. Genomic DNA of each strain was transferred onto positively charged nylon membranes (GE Healthcare) using a vacuum apparatus (Höefer vacuum system; GE Healthcare). The hybridization reactions were carried out with the ECL nucleic acid labeling and detection system (GE Healthcare) according to the instructions of the manufacturer.

Quantitative real-time RT-PCR.

Total RNAs of all aEPEC strains were obtained from three independent assays from cultures in DMEM and, after 6 h, adherence assays with HeLa cells. DMEM samples were obtained from aEPEC cultures in 30 ml of DMEM, inoculated with an LB broth preinoculum grown overnight (diluted at 1:100), and grown in a shaking incubator at 37°C until they reached the late exponential growth phase (optical density at 600 nm [OD₆₀₀] of 1.0). HeLa cell adherence samples were obtained by using HeLa cell monolayers in 75-cm² tissue culture flasks with 50 ml of DMEM containing 2% FBS and 1 ml of the LB broth preinoculum grown overnight. After 6 h of incubation, followed by PBS washings, the cultures were lysed with 3 ml of 1% Triton X-100. The entire volumes of both DMEM and HeLa cell adherence samples were centrifuged (4,000 × g for 30 min), and the pellets obtained were used for RNA extraction using the RiboPure bacterial RNA isolation kit (Ambion) according to the manufacturer's guidelines. The primers for the real-time PCR were designed by using Primer Express v 1.5 (Applied Biosystems), and the amplification efficiency and template specificity of each one of the primer pairs were validated as previously described (61). The real-time reverse transcription (RT)-PCR was a one-step reaction performed with an ABI 7500 sequence detection system (Applied Biosystems), and the reaction mixtures were prepared as previously described (61). Data were collected by using ABI Sequence Detection 1.3 software (Applied Biosystems). All data were normalized to the levels of rpoA (RNA polymerase subunit A) and analyzed by using the comparative threshold (C_T) method (61). The expression levels of the target genes under different culture conditions were compared by using the relative quantification method (2). Real-time data were expressed as changes of the levels of expression compared to the ones observed for BA320. Statistical significance was determined by a Student's t test, and a P value of ≤0.05 was considered significant.

Immunodetection of intimin, Tir, EspA, EspB, and EspD.

Total proteins from strains E2348/69, CVD206, EPEC Δtir, Ec292/84, 9100/83, BA320, and BA4013 (Table 1) were obtained from cultures aerobically grown in DMEM at 37°C until they reached an OD₆₀₀ of 1.0. Proteins were resolved by using SDS-PAGE with 12% polyacrylamide gels. After electrophoresis, separated proteins were transferred onto nitrocellulose membranes (GE Healthcare). The immunoblots were developed by employing anti-Tir (1:100) (12) and anti-intimin (1:100) (41) rabbit polyclonal antisera, followed by incubation with 1:5,000-diluted goat anti-rabbit alkaline phosphatase (Sigma). The reaction was revealed with 50 mg/ml each of nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP) (Promega). Secreted proteins from E2348/69, UMD872, UMD864, UMD870, Ec292/84, 9100/83, BA320, and BA4013 (Table 1) were obtained from cultures aerobically grown in DMEM at 37°C until they reached an OD₆₀₀ of 1.0, as previously described by Mairena et al. (37). Concentrated supernatants were resolved by using SDS-PAGE with 10% polyacrylamide gels and transferred onto polyvinylidene fluoride membranes (Millipore), as described above. The immunoblot was developed by employing rabbit polyclonal antisera against EspA (1:100), EspB (1:100), and EspD (1:100) (12), followed by incubation with 1:5,000-diluted goat anti-rabbit alkaline phosphatase (Sigma). The reaction was revealed with 50 mg/ml each of NBT and BCIP (Promega).

Introduction of an espF_U clone into aEPEC.

As espF_U and tccP refer to the same gene, in the present work we used the espF_U nomenclature (9). The aEPEC strains of this study were transformed with EspF_U-expressing plasmid pKC471, which is a low-copy-number plasmid that harbors the espF_U gene of EHEC strain EDL933 (9). pKC471 extraction was carried out with the Qiagen Plasmid Midi kit (Qiagen). Plasmid DNA was introduced into the aEPEC strains by the electroporation of competent cells, and transformants were selected on LB agar plates containing kanamycin. The presence of pKC471 was confirmed by the electrophoretic analysis of plasmid extractions of transformants. The aEPEC strains containing the EspF_U-expressing plasmid were named BA320(pKC471), Ec292/84(pKC471), 9100/83(pKC471), and BA4013(pKC471).

RESULTS

aEPEC strains maintain their adherence patterns and their respective characteristics in their abilities to cause A/E lesions in intestinal and nonintestinal epithelial cell lines.

Previously determined adherence patterns were confirmed with HEp-2 cells and evaluated with four additional cell lines of intestinal (Caco-2, T84, and HT29) and nonintestinal (HeLa) origins in order to check the influence of the epithelial cell origin on these phenotypes. All strains evaluated preserved their adherence patterns in all cell lines, i.e., LAL (strain BA320), AA (strain Ec292/84), DA (strain 9100/83), and NA (strain BA4013) (Table 2 and Fig. 1). The capacity to aggregate actin at the site of bacterial attachment, as an indicator of the A/E lesion, was also evaluated with all five different cell lines. As mentioned above, the aEPEC strains were previously classified as being FAS positive (BA320 and 9100/83) and FAS negative (Ec292/84 and BA4013) in HEp-2 assays (1, 5, 29). Similar results were observed after assays were performed with the different cell lines (Table 2 and Fig. 1).

Table 2.

Adherence, FAS, and immunofluorescence assays (6 h) of WT and WT(pKC471) strains performed with different cell lines cultivated in vitro^a

Strain	HeLa			HEp-2			Caco-2		T84		HT29
	Adherence pattern	FAS test result	IFL PT66 result	Adherence pattern	FAS test result	IFL PT66 result	Adherence pattern	FAS test result	Adherence pattern	FAS test result	Adherence pattern	FAS test result
BA320	LAL	+	−	LAL	+	−	LAL	+	LAL	+	LAL	+
BA320(pKC471)	LAL	+	−	LAL	+	−	LAL	+	LAL	+	LAL	+
Ec292/84	AA	−	−	AA	−	−	AA	−	AA	−	AA	−
Ec292/84(pKC471)	AA	−	−	AA	−	−	AA	−	AA	−	AA	−
9100/83	DA	+	−	DA	+	−	DA	+	DA	+	DA	+
9100/83(pKC471)	DA	+	−	DA	+	−	DA	+	DA	+	DA	+
BA4013	NA	−	−	NA	−	−	NA	−	NA	−	NA	−
BA4013(pKC471)	LAL	+	−	LAL	+	−	LAL	+	LAL	+	LAL	+
Controls
E2348/69	LA	+	+	LA	+	+	LA	+	LA	+	LA	+
DH5α	NA	−	−	NA	−	−	NA	−	NA	−	NA	−

^aIFL PT66, immunofluorescence assay for the detection of Tir phosphorylation; AA, aggregative adherence; DA, diffuse adherence; LA, localized adherence; LAL, localized adherence-like; NA, nonadherent.

Fig. 1.

Adherence assay and FAS test of wild-type aEPEC and aEPEC strains harboring pKC471 performed in 6 h using HeLa cells. Typical EPEC strain E2348/69 and E. coli strain DH5α were used as positive and negative adherence controls, respectively.

The LEE region is present and functional in aEPEC strains regardless of their capacity to cause A/E in vitro.

In order to examine the integrity of the LEE, the presence of all 31 LEE genes was verified by PCR using primers based on the LEE sequence of tEPEC strain E2348/69. The amplification of all 31 genes was obtained only with the AA strain, while the other three aEPEC strains exhibited negative results for at least one of the following 11 genes: escJ, cesF, espG, espB, escF, espH, sepQ, espD, cesD2, grlA, and espF (detailed results for each strain are presented in Table S2 in the supplemental material). However, these 11 genes were detected in all three PCR-negative strains by slot blot analysis of genomic DNA (Table S2).

In summary, the PCR and slot blot results indicated that the LEE region is present in all aEPEC strains studied, which directed us to evaluate the expression levels of LEE operons in these strains. To answer that, we sought to examine LEE transcriptional levels of all aEPEC strains using quantitative RT-PCR (qRT-PCR). The amounts of ler (LEE1), escC (LEE2), escV (LEE3), espA (LEE4), and eae (LEE5) were measured in two situations: at the late exponential growth phase in DMEM (LAL, AA, DA, and NA strains) and after bacterial contact with HeLa cells (LAL, AA, and DA strains), conditions known to activate LEE expression (35). For comparative analysis, the LEE transcriptional levels of the LAL strain (BA320) were used, since this strain represents a prototype of aEPEC; i.e., it expresses LAL adherence and has a positive FAS reaction after 6 h of interaction with epithelial cells (Fig. 2). The same comparative analysis using tEPEC strain E2348/69 as a reference can be found in Fig. S1 in the supplemental material.

Fig. 2.

Transcriptional profile of LEE operons of aEPEC strains. In experiments performed with DMEM and HeLa cells, transcriptional levels of ler (LEE1) (A and B, respectively), escC (LEE2) (C and D, respectively), escV (LEE3) (E and F, respectively), espA (LEE4) (G and H, respectively), and eae (LEE5) (I and J, respectively) were measured by real-time PCR. Relative fold expression represents the change (n-fold) in the transcriptional level compared to the level of LAL strain BA320 (gray bar, value of 1.0). Results are expressed as means and standard deviations of data from triplicate experiments. The levels of the rpoA transcript were used to normalize the C_T values to account for variations in bacterial numbers. Statistical significance was determined by a Student's t test based on comparisons with strain BA320 (*, P < 0.05; ⊠, P ≥ 0.05 [not significant]).

Transcriptional levels of ler (corresponding to the LEE1 operon) in AA, DA, and NA strains were significantly lower than the ones observed for the LAL strain (BA320) (P < 0.05) in the presence and absence of HeLa cells (Fig. 2A and B). In the AA strain and DA strain, transcriptional levels of ler were approximately 100-fold and 10-fold higher, respectively, in the assay with HeLa cells than in the cultures in DMEM. Similar results were observed for escC (LEE2) and escV (LEE3), where transcriptional levels were significantly decreased (P < 0.05) in both situations (Fig. 2C, D, E, and F), and for eae (LEE5) only in DMEM (Fig. 2I). On the other hand, there were no significant differences for eae transcription in the presence of HeLa cells (Fig. 2J). Comparing the results of assays performed with the adherent strains in DMEM and HeLa cells, we observed that, in comparison to the LAL strain (BA320) expression levels, the AA strain presented increased transcriptional levels of escC, eae (100-fold), and escV (10-fold); the DA strain presented increased transcriptional levels of escC and escV (5-fold) and no fold variation for eae.

Figures 2G and H show the transcriptional levels of LEE4. In comparison to the LAL strain (BA320), the AA strain (Ec292/84) presented a decreased transcriptional level in DMEM (P < 0.05) and in the presence of HeLa cells (P < 0.05), being 10-fold lower in the presence of the cells. The NA strain (BA4013) presented decreased transcriptional levels in DMEM (P < 0.05) and increased levels in HeLa cells (P = 0.002), and the value of the transcriptional level was 1 × 10⁶-fold higher in the presence of HeLa cells. The DA strain (9100/83) presented higher transcriptional levels of espA in DMEM (P < 0.05) and lower levels (100-fold) in the presence of HeLa cells (P < 0.05). The espA transcriptional levels of the AA and DA strains decreased when in contact with HeLa cells.

In addition to the analysis of LEE transcription levels, the expressions of EspA, EspB, EspD, intimin, and Tir were examined by immunoblotting. Figure 3 shows the different levels of expression of these five proteins in the aEPEC strain studied. The AA strain presented high expression levels of EspD and intimin and low levels of EspA, EspB, and Tir. The DA strain (9100/83) presented high expression levels of intimin, Tir, EspA, and EspD and low levels of EspB. The LAL strain (BA320) presented high levels of EspA, EspB, EspD, and intimin and low levels of Tir, and the NA strain presented an elevated level of expression of intimin, Tir, EspB, and EspD and low levels of EspA.

Fig. 3.

Expression of intimin, Tir, EspA, EspB, and EspD of aEPEC strains analyzed by immunoblotting. Typical EPEC strain E2348/69 was used as a positive control, and the following mutants were used as negative controls: CVD206 (eae mutant), EPEC Δtir (tir), UMD872 (espA mutant), UMD864 (espB mutant), and UMD870 (espD mutant). The whole-cell proteins intimin and Tir were separated on 12% SDS-PAGE gels. The secreted proteins EspA, EspB, and EspD were separated on 10% SDS-PAGE gels. The apparent molecular masses are indicated on the left.

EspF_U plays a role in adherence of aEPEC.

In order to evaluate if the FAS-positive strains (BA320 and 9100/83) employ the Nck pathway to establish A/E lesions in vitro, we examined if the Tir proteins of these strains were phosphorylated after translocation into HeLa and HEp-2 cells. The Tir proteins of these two strains were unable to be phosphorylated in the host cell membrane. As expected, the FAS-negative strains were also unable to phosphorylate Tir (Table 2). Therefore, none of the aEPEC strains used the Nck pathway in the formation of A/E lesions in vitro.

We also checked if the FAS-positive strains used the EspF_U pathway to establish A/E lesions by searching for the presence of espF_U. The LAL strain (BA320) was positive for espF_U using the tccP primers but negative with the tccP2 primers, while the other three aEPEC strains were negative for both tccP and tccP2 amplifications (see Table S2 in the supplemental material). Since the FAS-negative strains were devoid of espF_U, they were transformed with a plasmid that expresses EspF_U (9) in order to evaluate if the A/E-negative results were due to the lack of this protein. The FAS-positive strains (LAL and DA) were also transformed with this plasmid as controls. The transformed strains were then subjected to adherence and FAS assays with HeLa cells (Table 2 and Fig. 1).

The complemented AA [Ec292/84(pKC471)], DA [9100/83(pKC471)], and LAL [BA320(pKC471)] strains maintained their original adherence patterns in all cell lines, indicating no influence of EspF_U on the establishment of these phenotypes. On the other hand, BA4013(pKC471) displayed the LAL pattern and was also able to induce the A/E lesion in all cell lines after 6 h of incubation (Table 2 and Fig. 1).

The transcriptional levels of the LEE operons were also measured in strain BA4013(pKC471) in the presence of HeLa cells, since this strain was adherent. For comparative analysis, the LEE transcriptional levels of the LAL strain (BA320) were also used (Fig. 2B, D, F, H, and J). In summary, the results for LEE1, LEE2, LEE3, and LEE5 transcriptional levels were similar to those of the AA and DA strains in the presence of HeLa cells and always lower than those of the LAL strain. Interestingly, in BA4013(pKC471) the LEE4 transcriptional level was slightly higher than that of the LAL strain (Fig. 2H) and was 1 × 10⁶-fold in comparison to the transcriptional level of LEE4 in DMEM (Fig. 2G). Also, an immunoblotting assay showed that the expression levels of intimin, Tir, EspA, EspB, and EspD for BA4013(pKC471) were higher than those observed for wild-type (WT) strain BA4013 (Fig. 3).

DISCUSSION

aEPEC is a heterogeneous diarrheagenic E. coli subgroup in terms of serotypes, adherence patterns, and the presence of non-LEE virulence factors (25). The correct evaluation of the adherence pattern and the ability to cause A/E is important for the diagnosis and determination of aEPEC virulence. For this reason, we studied these characteristics of aEPEC strains displaying different types of interactions with cultured epithelial cells.

Previously observed adherence patterns of aEPEC strains in HEp-2 cells were maintained in other epithelial cells, including intestinal (Caco-2, T84, and HT29) and nonintestinal (HeLa and HEp-2) lines. However, the number of strains studied was too limited to conclude that the expression of these patterns is always equivalent in distinct cell lines. Indeed, Vieira et al. (58), who previously studied 59 aEPEC strains, demonstrated some variations in their adherence patterns in HeLa and Caco-2 cells. Also, Moreira et al. (43) previously showed variations of the adherence patterns of aEPEC strains belonging to serotype O51:H40 in HeLa, Caco-2, and T84 cells. These findings indicate that environmental and eukaryotic cell factors are stimulating the expressions of different adherence patterns. Among the strains analyzed in this study, AA and DA patterns are mediated by specific adhesins expressed by strains Ec292/84 and 9100/83, respectively (5, 29), and the LAL pattern of strain BA320 is probably mediated by intimin and EspA, as suggested by the analysis of a bundle-forming pilus (BFP) mutant of tEPEC strain E2348/69 (10). It is interesting that the incapacity of strain BA4013 to adhere to HEp-2 cells was also observed for intestinal cells, which resemble the human intestinal environment. Although Caco-2 cells express brush border enzymes more related to villi from small intestinal mucosa, all intestinal cells employed are derived from the human large intestine (44), and aEPEC may adhere better to the small intestine, as previously demonstrated for tEPEC (13). Nonadherent aEPEC strains have also been detected previously by other authors using cultured epithelial cells (24, 50, 52). The behavior of such strains in vivo remains to be evaluated.

Although our FAS results comparing different cell lines in vitro did not vary, other authors previously observed variations in the abilities to cause A/E lesions in different epithelial cells (43, 58), indicating that both cellular and bacterial factors are involved in the establishment of A/E lesions. For that reason, the FAS test should be considered with precaution, and in vitro organ culture (IVOC) of intestinal mucosa may represent a more suitable model to study the interactions of EPEC and EHEC with host cells (3, 19). The FAS-negative results for two strains in our study raised questions about the integrity of their LEE regions. The detection of some LEE genes was negative by PCR, although these same genes were detected by slot blot assays. These PCR-negative results were due to the lack of primer annealing, since they were based on the LEE sequence of tEPEC strain E2348/69. In fact, sequence analysis of some LEE genes of our aEPEC strains demonstrated a closer relationship of those sequences with LEE of EHEC O157:H7 strains (see Fig. S2 in the supplemental material), indicating the presence of slight differences between LEE sequences. Gärtner and Schmidt (23) showed previously that the LEE island is highly conserved, but not 100%, between aEPEC and tEPEC strains in a comparative analysis of LEE sequences of two DA aEPEC strains and tEPEC strain E2348/69.

Since the presence of all LEE genes was detected, the entire LEE region was presumably present in all aEPEC strains studied, which directed us to evaluate the expression levels of the LEE operons in these strains. The aEPEC strains evaluated presented a functional LEE region, regardless of their capacity to cause or not to cause A/E. Each strain showed different LEE expression levels, and these levels were higher in the presence of eukaryotic cells, confirming that host factors activate the LEE. For comparative analysis, the LEE transcriptional levels of the LAL strain (BA320) were used, since this strain represents a prototype of aEPEC; i.e., it expresses LAL adherence and a positive FAS reaction after 6 h of incubation. Prototype tEPEC strain E2348/69, on the other hand, expresses the LA adherence pattern and is FAS positive after 3 h of interaction with epithelial cells due to the presence of pEAF (35), which would interfere with the aEPEC strains in the comparative analysis of this study.

In summary, the transcription of all five LEE operons was detected; however, the majority of the genes presented lower levels of expression than those obtained for the LAL strain, except for espA (corresponding to LEE4). The EspA filament is formed by the polymerization of EspA protein monomers (reviewed in reference 20) during the infection, presenting a higher level of expression of this protein than those of other T3SS proteins. Leverton and Kaper (35) also verified previously that a significant increase in the level of transcription of espA occurred after 5 h of infection in the presence and absence of HEp-2 cells. Our results showed a high level of LEE expression under conditions of bacterium-cell contact (Fig. 2). These high levels in the presence of HeLa cells are probably due to the activation and/or induction of the LEE operons by HeLa cell components. Quorum sensing is involved in the LEE activation of tEPEC (55, 56), and it is possible that in our strains, this system is activating the LEE in a ler-dependent or -independent manner. Also, several other regulators that were not evaluated in this study, like BipA, IHF (integration host factor), FIS (factor for inversion stimulation), LexA, H-NS, PerC, GrlA, and GrlR, might directly or indirectly regulate the LEE in EPEC (reviewed in reference 39).

Among all LEE proteins evaluated, intimin was highly expressed in all strains. Due to the lack of pEAF in aEPEC, the characteristic adherence pattern and A/E lesion formation are evident only within 6 h of incubation, while the same can be observed within 3 h with tEPEC strains (25). In the AA strain, the EspA level was low, and therefore, its expression was not detected by immunoblotting. On the other hand, Barros et al. (5), who studied the same strain previously (Ec292/84), showed a high level of EspA expression by immunoblotting; however, it has to be pointed out that EspA was extracted after 18 h of incubation in DMEM, while in the present study EspA was extracted after 6 h. Taken together, these findings suggest that LEE expression levels might vary depending on the aEPEC strain used. Moreover, these results clearly demonstrate that the LEE is functional in all strains, including the FAS-negative strains.

The collected data concerning the expression of the LEE operons of the aEPEC strains of this work indicated that the inability to cause A/E lesions could be due to the host cell pathways involved in that cellular lesion. Therefore, we examined the machinery of the Nck and the EspF_U pathways in these strains. Since the Tir proteins of all aEPEC strains were not phosphorylated, none of the strains used the Nck pathway in vitro. As the LAL strain harbored the espF_U gene and was FAS positive, we believed that EspF_U was interacting with the IRSp53/IRTKS and/or N/WASP pathway in all the cell lines tested. Since the DA strain was FAS positive in all cell lines, there is a possibility that the espF_U gene of this strain was not detected by the primers used. Previous studies of tccP and tccP2 of EPEC strains reported variations in these gene sequences (22, 47). These results demonstrated that the LAL strain, and probably the DA strain, used the EspF_U pathway in vitro.

EspF_U presented no influence on either the establishment of adherence patterns or on the FAS results of the strains displaying LAL, AA, and DA patterns. On the other hand, the NA strain harboring espF_U displayed the LAL adherence pattern and was able to induce A/E lesions in vitro. The expression of the LAL pattern was probably a result of intimin/EspA-mediated adherence, which is similar to the resulting adherence displayed by a tEPEC strain mutated in BFP (10). Thus, EspF_U is activating eae and espA, via an unknown pathway, as demonstrated by the qRT-PCR and immunoblotting assays with the transformed NA strain. It is possible that the NA strain has lost the prophage harboring espF_U (9, 46) during its passage in the intestine and, consequently, its ability to adhere. Indeed, Bielaszewska et al. (6) previously described an EHEC O26:H11 strain that lost the stx gene, which is also located in a prophage, during passage through the human intestine. Therefore, it was shown that for the NA strain (BA4013), EspF_U plays a role in cell adherence. This activity of EspF_U was also reported previously by Ritchie et al. (49), who studied EHEC infection of rabbits and piglets, where EspF_U promoted bacterial association with the intestinal epithelium.

The expression of EspF_U in the NA strain led to the establishment of the A/E lesion in vitro, as a consequence of the adherent phenotype and subsequent translocation of Tir and EspF_U. On the other hand, the expression of EspF_U did not enable the AA strain to trigger actin polymerization in all cell lines tested. Interestingly, this finding differs from those reported previously by Bai et al. (3), who described an aEPEC strain of the same serotype (O125:H6) that used a pathway different from Nck and EspF_U to cause A/E lesions in vitro. This strain triggered A/E lesions only in an ex vivo model, and after being transformed with tccP or espF_U, only a small percentage of the adherent bacteria presented the ability to cause A/E lesions in assays using HeLa cells. Although belonging to the same serotype, these strains may present differences in tir sequences necessary for the activation of the EspF_U pathway (19). It is also possible that our strain (Ec292/84) causes A/E lesions only in vivo.

In summary, we demonstrate that aEPEC strains that are A/E negative in vitro (FAS negative) are potentially pathogenic, since they have all the attributes to cause A/E lesions in vivo. Therefore, the results of FAS tests performed with cultured epithelial cells should be considered with precaution, since this may not correspond to the actual capacity of aEPEC strains to induce A/E lesions. Also, the presence of EspF_U was sufficient to provide an adherent phenotype for a nonadherent aEPEC strain, possibly via a direct or an indirect activation of the LEE4 and LEE5 operons. The actual participation of EspF_U in such an activation is unclear but is currently under investigation by our group.