Nucleotide Sequence Analysis of the Enteropathogenic Escherichia coli Adherence Factor Plasmid pMAR7

Abstract

The complete nucleotide sequence was determined for pMAR7, an enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) plasmid that contains genes encoding a type IV attachment pilus (Bfp) and the global virulence regulator per. Prototypic EAF plasmid pMAR7 is self-transmissible, unlike the smaller EAF plasmid pB171, which has no genes encoding conjugative functions. The tra locus, a highly conserved 33-kb segment found in pMAR7, is similar to the tra (conjugation) region of the F plasmid. ISEc13 copies flanking the pMAR7 tra region could potentially mobilize or delete the tra genes. Hybridization of 134 EPEC strains showed that a complete tra region is present only in strains of the EPEC1 clonal group. This study confirms EPEC's potential for dissemination of virulence attributes by horizontal transfer of the EAF plasmid.Enteropathogenic Escherichia coli (EPEC) are an important cause of acute and persistent diarrhea in infants in developing countries. EPEC strains produce a characteristic intestinal histopathology called the attaching-and-effacing (A/E) lesion, which is characterized by intimate bacterial adherence to the epithelial membrane, effacement of the microvilli, and striking cytoskeletal changes (18). The A/E phenotype is encoded by a 35-kb chromosomal pathogenicity island (PAI) known as the locus of enterocyte effacement (LEE) (17). The LEE encodes a type III secretion system that translocates virulence effector proteins into host epithelial cells and the surface protein intimin, mediating intimate adherence to host cells (reviewed in reference 12). The A/E histopathology and the presence of LEE are also characteristic of other diarrheagenic strains, including enterohemorrhagic E. coli (EHEC).The large plasmid pMAR2, the first genetic element implicated in EPEC disease, is present in the prototypic EPEC strain E2348/69 (O127:H6) (2). Adherence of EPEC to HEp-2 epithelial cells and in vivo adherence to piglet intestinal epithelium was shown to be dependent on this self-transmissible plasmid. Transfer of this plasmid to a nonadherent E. coli K-12 strain conferred the ability to adhere to epithelial cells. Of 32 adherent strains with classical EPEC serotypes, 31 possessed similar large plasmids, subsequently named EAF for EPEC adherence factor plasmids. Adult volunteers developed diarrhea in nine of ten cases challenged with wild-type E2348/69 (13) but in only two of nine controls who ingested the plasmidcured derivative. Antibodies to an outer membrane protein expressed by the parent strain were detected after treatment with wild type but not the plasmid-cured derivative. Expression of the protein, later identified as intimin (11), was also linked to the plasmid. A 1-kb region of pMAR2, called the EAF probe, was the first molecular diagnostic tool for EPEC (19).The type IV pilus named the bundle-forming pilus (BFP) (8) encoded in a 14-gene locus, was the first virulence factor to be identified for the EAF plasmid (24, 25). The rope-like filaments of BFP connect EPEC bacteria into microcolonies i...

Pathogenomics of the Virulence Plasmids ofEscherichia coli

“…Within this plasmid was a cluster of genes encoding bundle-forming pili (bfp genes), which are responsible for the localized adherence patterns exhibited by EPEC, and the perABC genes, involved in the transcriptional activation of bfp and other chromosomally encoded virulence factors. The sequencing of a second plasmid, pMAR7, revealed a high degree of colinearity between pB171 and pMAR7 (19) (Fig. 2).…”

“…pMAR7 was derived from pMAR2 from EPEC strain E2348/69 belonging to the O127:H6 serotype. The primary differences between pMAR7/ pMAR2 and pB171 is the presence of an intact F transfer region in pMAR7/pMAR2, which is completely absent from pB171, and 16 ORFs in pB171 not present in pMAR7/ pMAR2, which are mostly mobile elements (19). pMAR2 was sequenced as a part of a recent genome sequencing effort involving strain E2438/69, and pMAR7 and pMAR2 were reported to be identical outside of three SNPs and two singlebase insertions/deletions within intergenic regions (79).…”

Molecular Evolution of Typical EnteropathogenicEscherichia coli: Clonal Analysis by Multilocus Sequence Typing and Virulence Gene Allelic Profiling

“…The entire bfpA gene was amplified and sequenced in 15 strains with STs for which bfpA allelic data were not previously available ( STs 4,5,7,8,11,15,16,17,and 18). Comparative sequence analysis revealed the existence of a 10th allele of bfpA, which we designated ␤7.1 (Fig.…”

“…Our results also indicate varying degrees of promiscuity among the different EAF types (Table 5). Recently, the complete sequence of a derivative of the wild-type EAF plasmid (pMAR7) from prototypical EPEC 1 strain E2348/69 was determined (11). In comparison to the EAF of O111:NM strain B171, the primary difference is the presence of the tra locus in pMAR7 (EPEC 1, EAF type 1) and its absence from pB171 (EPEC 2, EAF type 2).…”

“…The EAF plasmids from different EPEC strains show considerable variation in size (ϳ85 to 110 kb) (48) and, presumably, gene content. Comparison of the complete EAF plasmid sequences from two prototypical EPEC strains (O127:H6 EPEC 1 strain E2348/69 and O111:NM EPEC 2 strain B171) indicates that the EPEC 2 plasmid of B171 carries fewer genes (80 versus 115 open reading frames) and a greater percentage of intact or partial insertion sequence elements (33% versus 19%) than the pMAR7 plasmid of EPEC 1 strain E2348/69 (11,64). Nevertheless, certain parts of the plasmid show a high degree of sequence conservation among typical EPEC strains (47), particularly in the region encoding the bundle-forming pilus (BFP), a type IV fimbria whose production is associated with the LA phenotype.…”