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Experimental Biology and Medicine 228:370-376 (2003)
© 2003 Society for Experimental Biology and Medicine

SUPPLEMENT. FOOD SAFETY CONCERNS OF VEROTOXIN-PRODUCING ESCHERICHIA COLI

Description of a Novel Intimin Variant (Type {zeta}) in the Bovine O84:NM Verotoxin-Producing Escherichia coli Strain 537/89 and the Diagnostic Value of Intimin Typing

Joerg Jores1, Karen Zehmke, Juergen Eichberg, Leonid Rumer and Lothar H. Wieler

Institut für Mikrobiologie und Tierseuchen, Freie Universität Berlin, 10115 Berlin, Germany

Abstract

Infections with verotoxin-producing Escherichia coli (VTEC) has resulted in increasing numbers of human illnesses annually. These illnesses usually result from the ability of VTEC to cause the attaching and effacing lesions (AE lesion). The AE phenotype is encoded by the locus of enterocyte effacement (LEE) pathogenicity island. A key adhesion factor involved is the outer membrane protein intimin, encoded by the eae gene within the LEE. Intimin types {alpha}, ß, {gamma}, {delta}, and {varepsilon} have been described previously. Each intimin represents distinct phylogenetic lineages of LEE-positive strains. A new intimin type {zeta} was identified in a VTEC strain of the serotype O84:NM (nonmotile) that was isolated from a calf with diarrhea. {zeta} intimin showed the highest similarity (88%) of its amino acid sequence to the {alpha} intimin. For diagnostic purposes, we established a polymerase chain reaction (PCR) method for diagnosis of the key virulence traits of VTEC (i.e., verotoxins and intimins). This method also distinguishes between the toxins (VT1 and VT2) and the six intimin types. By applying the PCR method, intimin {zeta} in strains of other VTEC serotypes O84:H2, O92:NM, O119:H25, and O150:NM was identified. Because the intimin types represent distinctive phylogenetic E. coli lineages, application of the intimin subtyping PCR offers significant benefits. These include improving diagnosis of VTEC infection and increasing the understanding of evolution of attaching and effacing VTEC and other LEE-positive bacteria.

Key Words: Escherichia coli • intimin types • diagnosis • food-borne pathogens • verotoxins

Verotoxin-producing Escherichia coli (VTEC) are significant enteric pathogens responsible for hemorrhagic colitis and the hemolytic uremic syndrome in humans. It is well established that verotoxins (VT1 and VT2), encoded on lambdoid phages, are key virulence factors involved in VTEC infections (1–3). Recent epidemiological evidence suggests that the possession of additional virulence factors, namely the locus of enterocyte effacement (LEE) and the EHEC hemolysin (HlyEHEC), renders VTEC strains more virulent (2, 4). Although HlyEHEC can easily be monitored on washed blood agar dishes (5), identification of the LEE pathogenicity island requires more fastidious techniques. The LEE enables VTEC strains to cause the attaching and effacing (AE) lesions (6). The LEE-specific genes also are found in other bacteria such as enteropathogenic E. coli (EPEC), Citrobacter rodentium, and Escherichia alvei (formerly known as Hafnia alvei). The LEE encodes a type III secretion system, an adhesin (intimin) factor, the translocated intimin receptor (Tir), and at least five secreted proteins (7–9). The key adhesion factor intimin, an outer membrane protein, is encoded by a mosaic gene and consists of a conserved N-terminal region and a variable C-terminal region (9, 10). The C-terminal region of the protein was shown to be responsible for binding to the translocated intimin receptor (Tir) and the intimate binding activity of the outer membrane protein to eukaryotic enterocytes (11–13).

Five different intimin types have so far been identified, designated {alpha}, ß, {gamma}, {delta}, and {varepsilon} intimin. Types {alpha}, ß, {gamma}, and {delta} have been distinguished by antibodies, whereas type {varepsilon} was differentiated by polymerase chain reaction (PCR). Recent analysis by Oswald et al. (14) suggested that intimin {delta} is a subtype of intimin ß. Interestingly, the different intimin types are associated with the phylogeny of attaching and effacing E. coli strains (10, 14–17). Intimin {alpha} is specifically expressed by strains belonging to one evolutionary lineage of EPEC known as cluster EPEC 1. In contrast, intimin ß is mainly associated with strains of clusters EPEC 2 and STEC 2. Intimin {gamma} is associated with VTEC O157:H7 and EPEC O55:H7 (16, 18, 19). In addition to this important phylogenetic aspect, intimin types seem to be responsible for different host- and tissue-targeting specificities of the respective VTEC and EPEC strains (20, 21).

During the analysis of a larger set of LEE-positive bovine VTEC strains, we identified strains of O-types O4, O80, O84, O92, O119, and O150 with eae genes that were not typeable by PCR for differentiation of intimin types (14, 19). We hypothesized a novel intimin type, possibly being associated with a distinctive phylogenetic VTEC lineage. The objective was to describe the nucleotide sequence of the new intimin type, referred to as {zeta} intimin, in the bovine VTEC strain 537/89 of serotype O84:NM. Additionally, a diagnostic PCR for detection of intimin {zeta} was described.

Materials and Methods

Bacterial Strains.
Out of 122 bovine intimin-positive VTEC strains isolated from a total of 5438 E. coli strains (22), 36 representative strains were analyzed. Reference E. coli strains used for PCR and cell culture assays were previously reported (14, 23–28) and included PMK5 (EHEC O103:H2), MG1655 (E. coli K12), RW1374 (VTEC O103:H2), E2348/69 (EPEC O127:H6), EDL933 (EHEC O157:H7), 413/89-1 (VTEC O26:NM), and 537/89 (VTEC O84:NM), respectively.

DNA and PCR Analysis.
Total genomic DNA was isolated by using standard procedures (29). A total of 10 ng of template DNA was used for each PCR. Reactions were carried out in a total volume of 50 µl with Herkulase Enhanced Polymerase Blend (Stratagene, Amsterdam, The Netherlands) as recommended by the manufacturer. All PCR reactions were performed on a GeneAmp PCR System 2400 thermal cycler (Perkin-Elmer, Langen, Germany). Intimin {alpha} gene was detected by using a primer pair SK1-LP2, intimin ß by primer pair SK1-LP4, intimin {gamma} by primer pair SK1-LP3, and intimin {varepsilon} by primer pairs SK1-LP5 as described previously (14). The eae gene was tested by primers ECW1-ECW2 (30) as well as SK1-SK2 (14). The genes for VT1 and VT2 were identified (31) using primer pairs SK1 (VT) SK2 (VT) and SK3 (VT) SK4 (VT). The eae gene containing region in strain 537/89 was amplified (via PCR) by using primer pair eaef-escD1 (described herein). All primer sequences are given in Table IGo. DNA sequence analysis was performed by the chain termination sequencing technique (32). Phylogenetic analysis of eae sequences was performed by using the CLUSTAL W program (33). The {zeta} intimin gene sequence was deposited in GenBank (accession no. AJ298279).


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  		Table I. Sequence of Primer Pairs Used for Detection of Verotoxins and Intimins
 
Cell Culture Assays.
The ability of strain 537/89 to cause AE lesions was detected in HEp-2 cells and in fetal calf lung (FCL) cells by the fluorescence actin staining (FAS) test (34, 35).

Results

Types of eae Genes in Bovine VTEC Strains.
Out of the 122 eae-positive bovine VTEC strains described by Wieler et al. (22), the intimin types of 36 strains representing 26 serotypes were analyzed. By using the different eae primer pairs (Table IGo), four different intimin types in these 36 VTEC strains were identified. Intimin ß strains represented the largest group of strains (n = 17), and strains with this intimin type displayed the highest number (n = 14) of different serotypes. Intimin {gamma} was only identified in strains of serotypes O111:NM, O111:H2, O145:H28, and O157:H7, whereas the recently described intimin {varepsilon} was only found in strains of serotype O103:H2. Eight strains displaying different serotypes were not typeable by PCR identifying intimins of types {alpha}, ß, {gamma}, or {varepsilon}. These strains were O84:NM, O84:H2, O92:NM, O119:H25, O150:NM, O untypeable (OUT):NM, O4:NM, and O80:NM (Table IIGo).


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  		Table II. Distribution of Intimin Types Among Bovine VTEC Strains
 
Sequencing of the eae Gene of the Bovine VTEC Strain 537/89.
Because these eight strains harbored an eae gene (as tested with primer pairs SK1-SK2 and ECW1-ECW2) but were negative for other intimin types (14), the occurrence of a novel type of intimin was suspected. Therefore, the eae gene of strain 537/89 (O84:NM) was analyzed. This strain (isolated from a calf with diarrhea) contained the LEE pathogenicity island (data not shown) as indicated by hybridization experiments of genomic DNA with the LEE-specific probes A, B, C, and D (6). This strain also was able to induce a response in the FAS test using FCL cells, which indicated a functional intimin. However, it should be noted that no reaction with HEp-2 cells was detected. In addition, this strain harbored the VT1 gene as indicated by the PCR experiments (data not shown).

A PCR with primers located upstream and downstream of the eae gene was performed. The PCR with the primer pair eaef and escD revealed a product of 4 kb. Sequence analysis of the amplicon revealed an open reading frame of 2817 bp, which encodes a protein of 938 amino acids and has similarity to other intimin gene sequences. Two cysteine residues that have been shown to be necessary for the formation of a disulfide bond and the binding activity in other intimin types (9) were also found in the new intimin sequence (Fig. 1Go). The intimin of strain E2348/69 ({alpha} intimin) was shown to have the highest overall similarity (88%) to the intimin of 537/89 (data not shown). The C-terminal sequence (last 280 amino acids) was different from that of other intimin type sequences described so far and it had only 70% similarity to {alpha} intimin (Fig. 1Go). This sequence divergence indicated a new intimin type, referred to as intimin {zeta}. This finding prompted us to further investigate the set of bovine VTEC strains for the presence of this new intimin type.



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  		Figure 1. CLUSTAL W alignment of {psi} intimin from bovine VTEC strain 537/89 (O84:NM) with different published intimin types. This multiple alignment is based on the C-terminal amino acids of the displayed intimins starting with alanine 658. Different intimin sequences were retrieved from GenBank (VTEC 26:NM; AJ223063, Citrobacter rodentium; L11691, EPEC O127:H6; M58154, VTEC O157:H7; Z11541, E. alvei; L29509, VTEC O103:H2; AF116899, and VTEC O84:NM, AJ298279). Black shaded boxes indicate identity, gray shaded boxes indicate similarity of amino acids, and arrows indicate cysteine residues responsible for formation of disulfide bonds.

 
Construction of a Specific Intimin {zeta} Primer and Occurrence of the New Type Intimin in Other Bovine VTEC Strains.
On the basis of the {zeta} intimin gene sequence from strain 537/89, a specific reverse primer of the variable region of the 3' end (LP6), which could be used in combination with primer SK1, was generated. A PCR using primers SK1-LP6 yielded a product of the expected size of 2411 bp using chromosomal DNA from strain 537/89. In addition, the untypeable strains of serotypes O84:H2, O92:NM, O119:H25, and O150:NM were also positive in this PCR, revealing that they also harbored intimin {zeta} (Fig. 2Go). However, the same PCR did not generate amplicons with DNA from VTEC strains of serotypes OUT:NM, O4:NM, and O80:NM.



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  		Figure 2. PCR amplicons of different strains with primer pairs specific for intimin {alpha} (SK1-LP2), intimin ß (SK1-LP4), intimin {gamma} (SK1-LP3), intimin {varepsilon} (SK1-LP5), and intimin {zeta} (SK1-LP6). M, molecular weight marker ({lambda} HindIII); Lane 1 strain E2348/69 (O127:H6), Lane 2 strain 413/89-1 (O26:NM), Lane 3 strain EDL933 (O157:H2), Lane 4 strain PMK5 (O103:H2), Lane 5 strain 537/89 (O84:NM), Lane 6 strain IHIT3669 (O84:H2), Lane 7 strain IHIT3000 (O150:NM), and Lane 8 strain MG1655.

 
Phylogenetic Analysis of Intimin.
Based on the amino acid sequence of the new intimin variant, we constructed a phylogenetic tree to accumulate more information on the evolution of the intimin variants. Therefore, we performed a CLUSTAL W alignment by using the last 280 C-terminal amino acids of intimin (starting with alanine 656). In this analysis, intimin {zeta} was found to establish its own distinct branch, sharing highest phylogenetic relatedness with intimin {alpha} (Fig. 3Go).



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  		Figure 3. Phylogenetic tree based on five intimin types was generated using the C-terminal amino acids of the displayed intimins and starts with alanine 658.

 
Discussion

In addition to the five intimin types identified so far (14, 15, 36), we were able to characterize a novel type, termed intimin {zeta}. The biological significance of this finding has yet to be investigated, but it has previously been shown that the intimin type influences tissue and host tropism of attaching and effacing E. coli strains (20, 21). To our knowledge, VTEC strains of O-type O92 have so far only been isolated from cattle (22, 37–39). Clearly, VTEC strains of the three O-types O84, O92, and O150 are of low prevalence. Only strains of VTEC O84, O150, and O119:H25 have been isolated from cattle, sheep, or humans in Europe and in North America (22, 38, 40–42). Strains of O-type O119 traditionally represent classical EPEC strains (43, 44). Intimins of strains belonging to O-types O84, O92, O119, and O150 have not been characterized with regard to their respective intimin types {alpha}, ß, {gamma}, {delta}, or {varepsilon} (14, 19, 45). Moreover, the {zeta} intimin has been detected in strains of O-types O98, O111, and O156 (42, 46).

Although different intimin types are associated with distinctive phylogenetic lineages of LEE-positive E. coli strains (10, 15–17), our results do not suggest a {zeta} intimin adaptation to the bovine gut despite the finding that strain 537/89 reacted FAS positive when tested in bovine FCL cells and negative in HEp-2 cells of humans. However, the distribution of the {zeta} intimin in other LEE-positive E. coli strains remains to be elucidated.

The wide spectrum of different intimin types (ß, {gamma}, {varepsilon}, and {zeta}) identified in bovine VTEC disputes a species specific role of this adhesion factor. The finding that the intimins of serotype strains OUT:NM, O4:NM, and O80:NM could not be typed by PCR suggests a larger variety of intimins. These strains also may belong to subtypes of the distinctive intimins as described previously (14). Intimin {alpha} strains have exclusively been identified in EPEC (10). The identified variety of bovine intimin VTEC strains further substantiates the reservoir function of cattle for infections of humans with LEE-positive VTEC strains. It is not known why many phylogenetic different VTEC strains can be isolated from cattle. There is substantial knowledge of the transfer possibility of phages in the rumen (47), and it has been hypothesized that VT genes, encoded on phages, are spread in the ruminants intestines. This spread leads to the exchange of VT-encoding phages between E. coli in the intestines of ruminants (48). However, it should be noted that the natural transfer mechanisms of the LEE pathogenicity island is still unknown. Although we and others have identified remnants of phage sequences flanking different LEE pathogenicity islands, there is no indication of their functionality (7, 24).

It is possible that certain phylogenetic lineages are highly host adapted, and it has been suggested that the diversity within intimin is driven by natural selection (15). We were recently able to show that sera from calves reacted with different intensity when tested against recombinant intimin {alpha} and intimin ß proteins (49). It has also been reported that rabbit and human antisera react with differential intensity with intimin from different VTEC and EPEC strains (15, 50–52). Whether this difference has any protective value remains to be determined.

We are currently investigating the phylogeny of these {zeta} intimin strains in association with the clusters described by others (18). The phylogenetic investigation in Figure 3Go is based on the intimin sequence only. It revealed a distinct phylogenetic branch for this novel intimin type. The N-terminal region of the intimin {zeta} type showed the highest similarity of amino acids to type {alpha} intimin. Further research is needed to identify antigenic differences that may explain the biological properties of intimin {zeta} and that could be used to differentiate intimin types by the use of antibodies. Despite this missing antibody tool, we developed a PCR method to differentiate the new intimin in other LEE-harboring strains with specific primer pairs (SK1-LP6). With this approach, diagnostic laboratories should be able to screen LEE-positive E. coli strains and even strains of E. alvei or C. rodentium for their respective intimin types. Thus, by the application of PCR, investigators can better understand the virulence potential of these pathogens and can gain direct insight into the worldwide distribution and phylogeny of such VTEC strains.

Footnotes

This study was funded by the Deutsche Forschungsgemeinschaft Grant Wi 1436/3-2.

1 To whom request for reprints should be addressed at Institut für Mikrobiologie und Tierseuchen, Freie Universität Berlin, Philipstrasse 13, 10115 Berlin, Germany. E-mail: jores{at}zedat.fu-berlin.de Back

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