Cloning, Expression, and Characterization of Fimbrial Operon F9 from Enterohemorrhagic Escherichia coli O157:H7

Abstract

Recent transposon mutagenesis studies with two enterohemorrhagic Escherichia coli (EHEC) strains, a serotype O26:H-strain and a serotype O157:H7 strain, led to identification of a putative fimbrial operon that promotes colonization of young calves (1 to 2 weeks old). The distribution of the gene encoding the major fimbrial subunit present in O-island 61 of EHEC O157:H7 in a characterized set of 78 diarrheagenic E. coli strains was determined, and this gene was found in 87.2% of the strains and is therefore not an EHEC-specific region. The cluster was amplified by long-range PCR and cloned into the inducible expression vector pBAD18. Induced expression in E. coli K-12 led to production of fimbriae, as demonstrated by transmission electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The fimbriae were purified, and sera to the purified major subunit were raised and used to demonstrate expression from wild-type E. coli O157:H7 strains. Induced expression of the fimbriae, designated F9 fimbriae, was used to characterize binding to bovine epithelial cells, bovine gastrointestinal tissue explants, and extracellular matrix components. The fimbriae promoted increases in the levels of E. coli K-12 binding only to bovine epithelial cells. In contrast, induced expression of F9 fimbriae in E. coli O157:H7 significantly reduced adherence of the bacteria to bovine gastrointestinal explant tissue. This may have been due to physical hindrance of type III secretion-dependent attachment. The main F9 subunit gene was deleted in E. coli O157:H7, and the resulting mutant was compared with the wild-type strain for colonization in weaned cattle. While the shedding levels of the mutant were reduced, the animals were still colonized at the terminal rectum, indicating that the adhesin is not responsible for the rectal tropism observed but may contribute to colonization at other sites, as demonstrated previously with very young animals.Enterohemorrhagic Escherichia coli (EHEC) serotype O157:H7 causes gastrointestinal disease in humans that can be life threatening as a consequence of Shiga toxin activity on kidney and brain vasculature. Ruminants, particularly cattle, are the primary reservoir hosts for this organism (1, 3). Cattle are colonized without any overt symptoms and can shed EHEC O157:H7 in their feces for several weeks at levels up to 10 6 cells per g. Recent work has demonstrated that the predominant colonization site in cattle for E. coli O157:H7 is the final few centimeters of the terminal rectum, a site having a high density of lymphoid follicles and lymphoid-associated mucosa (19,21,30). A key aim of our research is to identify bacterial factors that drive this tropism for the terminal rectum.Considerable attention has been focused on type III secretion that leads to intimate attachment, and this has been shown to be important for ruminant colonization in a number of studies (5,7,8,22,44). For example, in a recent signaturetagged mutagenesis (STM) screening for genes that promote E. col...

Intestinal adherence associated with type IV pili of enterohemorrhagic Escherichia coli O157:H7

“…Several fimbriae have been identified in EHEC strains, including the sorbitolfermenting EHEC O157 fimbriae plasmid-encoded (SFP) (29), 2 loci-encoding long polar fimbriae (30), curli (31), F9 (a type I pilus homolog) (32), and a type IV pilus (TFP) in LEE-negative non-O157: H7 STEC (33). The role of most of these pili in host colonization is unknown.…”

Expression of the Escherichia coli IrgA homolog adhesin is regulated by the ferric uptake regulation protein

“…However, the mechanism of initial adherence of these organisms is not well understood. Candidate EHEC adherence factors include long polar fimbriae (LPF) related to those of Salmonella enterica [2,3]; ToxB, encoded on plasmid pO157, which positively influences the adherence of O157:H7 [4]; EHEC factor for adherence (efa1) [5] in sorbitol-fermenting O157:H-(but which is not found in the closely related E. coli O157:H7); an sfp operon homologous to the P-fimbrial operon of uropathogenic E. coli also found in sorbitol-fermenting O157:H- [6]; and F9 fimbriae [7].…”

Commensal and pathogenic Escherichia coli use a common pilus adherence factor for epithelial cell colonization

“…Intimin is an outer-membrane protein encoded on the LEE that mediates close adherence to the enterocyte cell membrane (23) via its own translocated receptor (Tir) (24), integrin (25), or a host cell protein called nucleolin (26). Other less well characterized surface adhesins have been reported (27)(28)(29)(30)(31), but their role in in vivo adherence and host colonization remains elusive. The genome of EHEC O157:H7 strains contains 16 loci-encoding genes putatively involved in pili biosynthesis (6,8,32); however, what role they play in the pathogenic scheme of these organisms remains largely unknown.…”