Tissue Tropism of Enteropathogenic Escherichia coli Strains Belonging to the O55 Serogroup

Abstract

Four enteropathogenic Escherichia coli (EPEC) strains belonging to the O55 serogroup (G21 and G30 [both O55:H6], G35 [O55:H−], and G58 [O55:H7]) were tested for their tissue tropism by using human intestinal in vitro organ culture. Strains showed restricted adhesion with attaching-and-effacing activity to follicle-associated epithelium of Peyer's patches, with no apparent adhesion to duodenum or colon. G35 and G58 express intimin γ and show a similar tropism to intimin γ-expressing enterohemorrhagic E. coli (EHEC) O157:H7. However, strains G21 and G30 were unusual because they expressed intimin α and had a restricted tissue tropism of intimin γ phenotype. The amino acid sequence of the carboxy-terminal 280 amino acids of intimin from G21 was determined. Comparison with the prototype intimin α from strain E2348/69 (O127:H6) showed a single amino acid difference (corresponding to Val907 and Ala907 in the whole intimins). This mutation was reproduced by site-directed mutagenesis in an intimin α plasmid template, pCVD438, with the hypothesis that it may induce a change in tropism. However, when the mutated plasmid was placed in both EPEC and EHEC backgrounds, duodenal adhesion in a manner similar to strain E2348/69 was evident upon in vitro organ culture. Thus, additional factor(s) unrelated to intimin exist in the O55:H6 genome that influence human intestinal tissue tropism.

Enteropathogenic Escherichia coli (EPEC) are an important cause of acute and prolonged diarrhea in the developing world with between 30 to 40% of diarrhea in the first year of life being attributed to EPEC (31).

E. coli is a clonal species, with clones identified on a combination of O and H antigens. The O antigen is part of the lipopolysaccharide present in the outer membrane of gram-negative bacteria (42, 45). E. coli serogroup O55 is the third most frequent EPEC O serogroup involved in infantile diarrhea, and O55:H6, O55:H7, and O55:H− account for 90% of the strains isolated (37). EPEC and enterohemorrhagic E. coli (EHEC) can be divided into two major groups of related clones, designated EPEC clones 1 and 2 and EHEC clones 1 and 2. EPEC clone 1 typically expresses flagellar antigen H6, whereas EPEC clone 2 expresses flagellar antigens H2 or H−. EHEC clone 1 includes the O157:H7 serogroup, whereas EHEC clone 2 contains other Shiga toxin-positive E. coli serogroups. In addition, EHEC strains belonging to clone 2 are closely related to the typical EPEC strains, whereas EHEC O157:H7 is related to the atypical EPEC O55:H7 (12).

Typical EPEC strains are classified as being positive for gene probes to eae (encoding intimin), EAF (EPEC adherence factor plasmid), and bfpA (encoding bundlin, the subunit pillin of the bundle-forming pilus) (20). The O55:H− strains are considered to have lost their flagella by mutation, since the nonmotile derivatives and the motile progenitor usually have the same biochemical properties. Therefore, it has been suggested that H− strains of the O55 serogroup are derived from H6 or H7 strains (37).

EPEC and EHEC have the ability to produce attaching-and-effacing (A/E) lesions on enterocytes (30, 35, 44). The A/E lesion is characterized by localized destruction of the brush border microvilli with intimate attachment of the bacteria to the enterocyte. The area underlying the bacteria consists of polymerized actin, ezrin, talin, and myosin (14) and the actin-regulating proteins WASP (Wiskott-Aldrich syndrome family of proteins) and Arp 2/3 (actin-related proteins 2 and 3 complex) (19). This host cell-bacterium interface may be raised above the host cell surface producing a pedestal. The genes necessary and sufficient for the production of an A/E lesion by EPEC are located on a pathogenicity island termed the locus of enterocyte effacement (LEE) (28). EHEC and other A/E bacteria, such as Citrobacter rodentium, also contain the LEE (4, 27) but in EHEC the LEE is necessary but not sufficient for A/E lesion production (8). Intimin, a 94- to 97-kDa protein encoded by the eae gene within the LEE, is an adherence factor, which has been shown to play a key role in intestinal colonization (12). Intimin mediates intimate attachment to the epithelial cell by binding to another LEE-encoded protein, the translocated intimin receptor (Tir), which is delivered into the host cell by a type III secretion system (22). The importance of intimin in human disease is highlighted by an eae mutant being significantly attenuated in human volunteers compared to the wild-type parent strain (7) and by the presence of a high titer of serum intimin antibodies in both individuals infected with EHEC (18) and in the colostrum of mothers in Brazil where EPEC infection is endemic (25).

To date, five intimin types have been identified and designated α, β, γ, δ, and ɛ (1, 32). Intimin α and intimin γ are associated with different evolutionary branches of EPEC and EHEC. Intimin α is associated with EPEC branch 1 and intimin γ is specifically associated with EHEC O157:H7 and the related EPEC O55:H7 (46). Intimin exchange studies in piglets have shown that varying the intimin type can alter the site of intestinal colonization by EHEC (43). This activity of intimin was further characterized by using in vitro organ culture (IVOC). The prototype intimin α-expressing strain O127:H6 E2348/69 produces A/E lesions on duodenum, terminal ileum, Peyer's patches and, in a small percentage of incubations, on the colon (33). In contrast, intimin γ-expressing EHEC exhibited a restricted tropism toward the follicle-associated epithelium (FAE) of Peyer's patches (35). When intimin γ from O157:H7 was placed in an eae mutant strain of E2348/69, adherence to the FAE predominated, thus mimicking the restricted tissue tropism of O157:H7 (35). In contrast, placing intimin α in an eae mutant strain of O157:H7 resulted in colonization of the small intestine, as well as Peyer's patches (9). Thus, intimin is able to modulate the tissue tropism of EPEC and EHEC on human intestinal explants, producing characteristic intimin α- and γ-related phenotypes.

The receptor binding activity of intimin has been localized to the C-terminal 280 amino acids (Int280) (10). The global fold of Int280α was determined by nuclear magnetic resonance (21) and crystallography (26), which showed that it is built from three globular domains with the first two comprising β-sheet sandwiches. The third C-terminal domain (residues 183 to 280) has a topology that resembles C-type lectin domains (CTLD), a family of proteins responsible for cell surface carbohydrate recognition. Analysis of intimin-Tir complexes revealed amino acids likely to be involved in binding (3, 26) and showed these residues to be concentrated in a solvent-exposed region of the CTLD. Site-directed mutagenesis of residues within the CTLD was able to modulate the biological activity of intimin; in particular, substituting a valine at 911 with alanine in intimin α produced a derivative, which bound to intestinal mucosa in an intimin γ-like phenotype (36).

We studied the EPEC O55 serogroup, which expresses different intimin types, by using in vitro organ culture to further elucidate the mechanisms involved in tissue tropism.

MATERIALS AND METHODS

Bacterial strains and plasmids.

Bacterial strains and plasmids used in this study are listed in Table 1. Strains and plasmids were grown overnight at 37°C with shaking (250 rpm) in L broth (LB; Sigma) for DNA sequencing and site-directed mutagenesis; for infection assays, bacterial strains were grown overnight without shaking in brain heart infusion broth.

TABLE 1.

List of strains and plasmids used during this study

Strain or plasmid	Description	Source or reference
XL1-Blue	EndA -ve competent E. coli K-12 strain	Stratagene
CVD206	eae deletion mutant of O126:H7 strain E2348/69	5
ICC170	eae deletion mutant of O157:H7 strain 85/170	9
G21	Intimin α, EAF+, Bfp+, O55:H6	1, 37
G30	Intimin α, EAF+, Bfp+, O55:H6	1, 37
G35	Intimin γ, EAF+, Bfp+, O55:H−	1, 37
G58	Intimin γ, EAF−, Bfp+, O55:H7	1, 37
pCVD438	pACYC184 encoding intimin α	5
pCPG1	pMAL-c2 encoding intimin αO55:H6	This study
pCPG2	pACYC184 encoding intimin αA907V	This study

Human intestinal in vitro organ culture adhesion assay.

Tissue was obtained with fully informed parental consent and local ethical committee approval by using grasp forceps during routine endoscopic (Olympus PCF pediatric endoscope) investigation of intestinal disorders. Duodenal, terminal ileal, Peyer's patch, and transverse colonic biopsies were taken from areas showing no macroscopic abnormalities. Light microscopy subsequently showed no histological abnormality. IVOC infections were performed as described previously (15). Briefly, 25 μl of an overnight bacterial culture was inoculated onto the biopsy samples from the various regions of the gut. The IVOC medium was changed every 2 h, and the assay was terminated at 8 h. Each bacterial strain was examined on several occasions by using tissue from different patients. Samples were fixed with 2.5% glutaraldehyde and postfixed in 1% aqueous osmium tetroxide and viewed by scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

DNA sequencing of intimin from O55:H6 strain G21.

Genomic DNA from G21 was isolated by using the DNAeasy tissue kit (Qiagen), according to manufacturer's instructions. The 3′ end of the eae gene encoding Int280α from O55:H6 was amplified by PCR by using high-fidelity Pfu DNA polymerase (1 cycle of 94°C for 1 min, followed by 25 cycles of 94°C for 30 s, 50°C for 1 min, and 74°C for 1 min, followed by 1 cycle of 74°C for 5 min; forward primer, 5′-GGAATTCATTACTGAGATTAAGGCT-3′; reverse primer, 5′-CGGGATCCTTATTTTACACAAGTGGC-3′). The amplified DNA and the plasmid pMAL-c2 were cut by using the restriction enzymes EcoRI and BamHI. The amplified DNA was inserted into cut pMAL-c2 by ligation with T4 DNA ligase. pMAL-c2 was transformed into E. coli XL1-Blue cells. Ampicillin resistant transformants were randomly selected and screened for the appropriate insert. XL1-Blue colonies containing the recombinant plasmid were grown overnight in LB, the plasmids isolated by using the plasmid miniprep kit (Qiagen) and ethanol precipitation. DNA sequencing was performed with 1 to 2 μg of plasmid DNA, and the Int280 forward and reverse primers (36).

Site-directed mutagenesis.

Site-directed mutagenesis of intimin α was performed by using the QuickChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Plasmid pCVD438 encoding intimin α (5) was used as a template. A complementary mutagenesis oligonucleotide pair incorporating a single amino acid substitution was designed. Primers were as follows: forward primer (5′-CAGCTCAAGATGTGAAGAGTGGTGTTGC-3′) and reverse primer (5′-GCAACACCACTCTTCACATCTTGAGCTG-3′).

Mutated plasmid containing staggered nicks was generated by extension of primers annealed to opposite strands of the denatured plasmid by temperature cycling in the presence of the high-fidelity Pfu DNA polymerase. Synthesized DNA containing the desired mutation was selected from the original DNA template by incubation with the enzyme DpnI at 37°C for 1 h, which cleaves the parental DNA at dam methylated GATC sequences, leaving the unmethylated newly synthesized mutated plasmid intact. Nicks in the plasmid were repaired after transformation of 1 μl of the synthesized DNA into competent E. coli XL1-Blue cells. Chloramphenicol-resistant transformants were randomly selected and grown overnight in LB. Plasmid DNA was isolated by using the plasmid miniprep kit (Qiagen) and ethanol precipitation. Correct incorporation of the mutation was monitored by DNA sequencing by using an automated DNA sequencer. The mutated plasmids were then transformed into eae deletion mutants of EPEC strain CVD206 (5) and EHEC strain ICC170 (9).

RESULTS

IVOC tissue tropism of O55 serogroup strains G21, G30, G35, and G58.

Intimin, from EPEC and EHEC, is essential for A/E lesion formation in IVOC (9, 15). All of the EPEC strains expressed intimin, as shown by Western blotting with a universal intimin antiserum (2) (data not shown). SEM of the uninoculated controls showed good preservation of mucosal architecture, the epithelium was intact and there was no evidence of excess extrusion of enterocytes or mucus. All O55 strains showed a restricted pattern of A/E lesion formation, which was limited to FAE overlying ileal Peyer's patches (Fig. 1 and Table 2). Thus, the intimin γ-expressing strains G35 (O55:H−) and G58 (O55:H7) showed the same tissue tropism as EHEC O157:H7 (35), which expresses intimin γ. Strains expressing intimin α can produce A/E lesions on duodenum and ileum, as well as Peyer's patches (9, 33). However, O55:H6 strain G21, which expresses intimin α, did not reproduce this phenotype, forming A/E lesions on Peyer's patches alone. In order to investigate if this phenomenon is unique to strain G21 or common to other O55:H6 strains, a second isolate, strain G30 was tested in IVOC. Like G21, strain G30 did not adhere to duodenum, terminal ileum, or colon. These results indicate that intimin α strains may show a variable pattern of tissue tropism.

FIG. 1.

SEM of IVOC of O55 Strains. (A) Low-power view of lymphoid follicles within IVOC sample from ileal Peyer's patch region. (B to D) O55:H6 (B), O55:H− (C), and O55:H7 (D), showing adhesion of strains to the FAE of an ileal Peyer's patch. Bars: 100 μm (A), 0.5 μm (B and C), 5 μm (D).

TABLE 2.

Patient age and number of biopsies with adherent bacteria expressed as a proportion of biopsies inoculated

Strain	No. of biopsies with adherent bacteria/no. of biopsies inoculateda
	Small intestine	Peyer's patch	Colon
G21	0/4	2/3	0/3
G30	0/3	ND	0/3
G35	0/4	4/4	0/3
G58	0/7	3/3	0/3
CVD206	0/5	ND	ND
CVD206(pCPG2)	5/5	ND	ND
ICC170(pCPG2)	3/3	ND	ND

^aThe median patient ages (in months) for each group were as follows: small intestine, 115 (range, 9 to 188); Peyer's patch, 73 (range, 30 to 171); and colon, 119 (range, 103 to 167). ND, not done.

TEM was used to confirm the presence of A/E lesions on intestinal Peyer's patch explants (Fig. 2). In all three O55 strains, intimate adherence of bacteria to the enterocyte with effacement of microvilli could be seen. The bacterium-enterocyte interface was primarily seen as a depression, with only a few pedestal-like structures observed. No bacteria were observed within enterocytes.

FIG. 2.

TEM of IVOC of O55 Strains. (A to C) O55:H6 (A), O55:H− (B), and O55:H7 (C), showing A/E lesions on the FAE of an ileal Peyer's patch. Note the lack of pedestal formation. Bar, 0.5 μm.

Sequencing of intimin α from O55:H6 strain G21.

Site-directed mutagenesis within the CTLD region of intimin α has shown that the mutation of a specific residue (Val911) can lead to the production of an intimin α-expressing EPEC derivative with a restricted colonization phenotype (36). To determine whether the eae gene of G21 contained any variation in this region, the carboxy-terminal region encoding Int280 was compared to that of the prototype intimin α from O127:H6 strain E2348/69. This revealed a single amino acid change at position 907 (Fig. 3), i.e., a valine residue in G21 instead of an alanine residue as in E2384/69. In order to assess the influence of the presence of valine at position 907 in the intimin α of G21, site-directed mutagenesis was performed on pCVD438, which contains eaeα from E2348/69, generating plasmid pCPG2. The plasmid was transformed into the eae mutant E2348/69 strain CVD206 and the eae mutant O157:H7 85/170 strain ICC170. These strains, CVD206(pCPG2) and ICC170(pCPG2), were tested by using the IVOC assay.

FIG. 3.

Comparison of carboxy-terminal amino acid sequences of intimin α from E2348/69 (AAC 38392) and G21 (O55:H6). Amino acids are numbered according to the position in intimin α of E2348/69.

IVOC assay of CVD206(pCPG2) and ICC170(pCPG2).

CVD206(pCPG2) was tested for A/E lesion formation on duodenum by using the IVOC assay. It was important to use duodenal explants as A/E lesion formation on the proximal small intestine is a reported characteristic of intimin α strains, whereas A/E lesion formation on FAE of Peyer's patches is synonymous with strains expressing intimin α and with strains expressing intimin γ. CVD206 showed no adhesion (Table 2) as reported earlier in qualitative form. CVD206(pCPG2) produced A/E lesions on the duodenum in a manner similar to that of E2348/69 (Table 2 and Fig. 4A). This indicates that the difference in intimin amino acid sequence between G21 and E2348/69 cannot be the determining factor in the restricted tissue tropism of G21.

FIG. 4.

SEM of IVOC. (A and B) CVD206(pCPG2) (A) and ICC170(pCPG2) (B), both showing A/E lesion formation on the duodenum. Bar, 0.5 μm.

The use of the EPEC derived strain CVD206 as a recipient, for pCPG2, may have been a factor in the inability of CVD206(pCPG2) to reproduce the restricted phenotype associated with G21. To test this, pCPG2 was placed in ICC170, an eae mutant strain derived from EHEC 85/170 which only produces A/E lesions on follicle-associated epithelium (9). The resultant strain, ICC170(pCPG2), produced A/E lesions on duodenum in a manner similar to that of CVD206(pCPG2) (Table 2 and Fig. 4B). Therefore, the restricted tissue tropism of O55:H6 G21 is associated with a factor distinct from intimin, which is not in the EPEC E2348/69 or the EHEC 85-170 backgrounds.

DISCUSSION

The E. coli O55 serogroup is a prominent cause of infantile diarrhea. Each EPEC serogroup is made up of clones with distinct combinations of virulence factors. In the case of the O55 serogroup this results in some strains belonging to the classic EPEC genotype and others having an atypical EPEC genotype with close links to EHEC O157:H7. The similarity between O55:H7 and O157:H7 is demonstrated by their nearly identical sequences for the H7 flagellin gene and the similarity of their eae alleles (29, 37). The eae gene encodes the outer membrane protein intimin, which allows EPEC and EHEC to adhere to human intestinal cells and produce A/E lesions (9, 15). This protein can be divided into three globular domains. The CTLD, at the carboxy terminus of intimin, is responsible for binding of intimin to its translocated receptor, Tir. The 280-amino-acid carboxy-terminal region also allows intimin to induce microvillus-like processes on Hep-2 cells (34), acts as an immune modulator (16), and may bind a host cell receptor (12). Intimin α has been show to bind β1 integrin in vitro (11), although this process is not essential for A/E lesion formation (23). In addition, intimin γ binds nucleolin, which may act as a host-derived cell surface receptor during the initial stages of EHEC adhesion (40).

Intimin can be divided according to antigenicity, and so far five distinct subtypes have been published (1, 32). The significance of the different subtypes in pathogenesis remains to be fully elucidated. Recent studies have shown a correlation between intimin type and tissue tropism (9, 33). The data showed that intimin α and intimin γ produce different tropism phenotypes in a manner that is Tir type independent. Using IVOC, intimin α has been shown to allow both EPEC and EHEC to produce A/E lesions on duodenum and Peyer's patches, whereas intimin γ limits A/E lesion formation to Peyer's patches.

In this study we have shown that EPEC strains from the O55 serogroup, expressing intimin α or intimin γ, adhered preferentially to the FAE of Peyer's patches. In the case of O55:H6 strains G21 and G30, this is the first example of intimin α-expressing strains showing the lack of adherence to the small intestinal villous surface.

A recent study of the biological activity of intimin α highlighted certain important residues involved in intimin-Tir interaction (36). The valine at position 911 is hydrophobic and lies at the top end of the Tir-binding site. It is solvent exposed and as such is ideally placed to interact with Tir (21). Its mutation to an alanine residue resulted in restricted tissue tropism, with A/E lesion formation being limited to the FAE of Peyer's patches. An equivalent alanine substitution at position 906 in intimin γ led to disruption of Tir-binding activity (24). Thus, the removal of a hydrophobic valine residue, within the Tir-binding site of intimin α and intimin γ, and substitution with alanine led to altered binding activity and in the case of intimin α, restricted A/E lesion formation in IVOC. The intimin α of G21 was sequenced and showed a single amino acid mutation at position 907 compared to the intimin α of O127:H6 strain E2348/69. Using the intimin α template plasmid, pCVD438, we constructed an intimin α with a valine at position 907, thus matching G21. When this plasmid (pCPG2) was placed in an EPEC or an EHEC background, it showed a typical intimin α phenotype and adhered to duodenum. Thus, the mutation at position 907 was not a factor involved in the restricted adherence of G21 to FAE of Peyer's patches. It is theoretically possible that residues outside the C-terminal domain of intimin are involved in the determination of tissue tropism, but these residues show >90% homology between the various intimin types, so it remains a remote possibility. It is more likely that the restricted tissue tropism of G21 is determined by other genes which are specific to the O55:H6 genome. Several different infection models have shown that intimin is involved in tissue tropism (9, 33, 36, 43) and thus it appears that an O55:H6 factor is operating to prevent colonization of small intestinal villous surfaces despite the possession of intimin α. If this factor can be identified it will be interesting to see whether it is also present in the intimin γ-positive O55:H−, O55:H7, and O157:H7 strains, which would suggest a multifactorial control of restricted adhesion to the FAE of Peyer's patches.

The restricted adhesion of strain G35 (O55:H−) to Peyer's patches is in line with the fact that it expresses intimin γ, although it also possesses the EAF plasmid (37) and hence the genes for BFP production (13), i.e., it is a typical EPEC (20). Thus, the presence of BFP, which has been considered to be an initial adhesion for EPEC colonization (6), is not synonymous with villous adhesion. Indeed, in a previous study with IVOC, BFP was suggested to be involved in three-dimensional colony formation and not initial adherence (15). Such a role would remain compatible with adhesion restricted to the FAE, i.e., to establish colony units within the gut environment, allowing nonintimate bacterium-to-bacterium adhesion and, once a quorum of bacteria have been established, to permit disaggregation and colonization of other regions of the gut.

TEM showed limited evidence of pedestals (19, 38) being formed by the three O55 strains. Rather, the intimate adhesion between bacterium and host cell membrane resulted, generally, in slight depressions in the surface of the enterocyte. A similar appearance was shown in newborn pigs infected with O55:H7 (41), indicating it is not just due to the relatively short-term length of incubation in IVOC. Some pedestal-like structures were illustrated, but these were located on extruding cells where cytoplasmic extrusion gives the appearance of elongated pedestals (41). In addition, pedestal formation in in vivo EPEC infections (17, 39, 44) and in animal models of human EPEC infection (30) are similar to those produced by IVOC (15). The lack of elongated pedestal or pseudopod (38) formation on gut explants highlights the difference between IVOC and cell line adhesion studies. Nonpolarized epithelial cells, derived from the larynx, lung, and cervix do not possess tight junctions, lack a terminal web, and do not have the complex actin-based cytoskeleton of the intestinal brush border. Thus, actin polymerization at the bacterium-host interface may be a more tightly regulated process in gut epithelium than in cell lines.

Strain O55:H7 was shown to enter the enterocytes of newborn pigs, implying an invasive potential (41). This phenomenon was not apparent after 8 h IVOC with Peyer's patch explants and has not been reported in other, non-O55 serogroup, in vivo EPEC infections in humans.

In summary, EPEC strains from the O55 serogroup show initial adherence to the FAE overlying Peyer's patches. The mechanism of this tissue tropism includes intimin and other factor(s) specific to the O55 genome. Further analysis of EPEC and EHEC genomes will allow us to determine the mechanisms by which these pathogens adhere, maintain their presence, and spread within the gut.