Characterization of the Adhesin of Escherichia coli F18 Fimbriae

Abstract

Previous research has suggested that the adhesin encoded by the F18 fimbrial operon in Escherichia coli is either the FedE or FedF protein. In this work, we show that anti-FedF antibodies, unlike anti-FedE serum, were able to inhibit E. coli adhesion to porcine enterocytes. Moreover, specific adhesion to enterocytes was shown with purified FedF-maltose binding protein.The operons of many fimbrial adhesins of Escherichia coli are well characterized (4). They contain genes coding for the major subunit protein, molecular chaperone and usher proteins, minor subunits, adhesin, and proteins of unknown function (4,11,12). The genes involved in the biosynthesis of F18 fimbria have been only partially described (5, 6). The major protein of the F18 fimbria, FedA, is not sufficient for recognizing the F18 receptor (5). Two additional genes from the fed gene cluster, fedE and fedF, have been described as essential for fimbrial adhesion and fimbrial length (6). However, so far it has not been possible to assess F18 adhesion function with regard to either of the two gene products.In this study, we sequenced the unknown region of the E. coli fed gene cluster and produced and purified FedF and FedE as fusion proteins with maltose binding protein (MBP) for raising antisera for adhesion studies. Furthermore, using indirect immunofluorescence microscopy and adhesion inhibition tests, we have characterized the FedF proteins as the adhesin of F18 fimbriae.Sequencing of the plasmid pIH120. The entire gene cluster encoding E. coli F18 fimbria was sequenced from the plasmid pIH120 (6) with an ABI 310 sequencer according to the manual of the manufacturer (PE Applied Biosystems). pIH120 was transferred into an E. coli HB101 host, resulting in strain ERF2055. Sequence analyses revealed that the fed gene cluster is composed of five genes. The gene coding for the major protein of F18 fimbria (fedA) and the genes encoding two minor proteins (fedE and fedF) were described earlier (5, 6). Two additional open reading frames were found between fedA and fedE and were designated fedB and fedC. FedB showed the highest similarity (83% identity) to the AfrB protein (AAC28316) from E. coli RDEC-1 (Fig. 1) and significant homology to other usher proteins involved in the biosynthesis of microbial pili (3). The second open reading frame (fedC) overlapped the 3Ј end of fedB, and its product had high identity (82%) with the periplasmic chaperone AfrC (AAC228317) from E. coli RDEC-1. Both FedB and FedC possess a predicted signal peptide for transmembrane secretion with a putative cleavage site for a signal peptidase between amino acids 23 and 24. The calculated molecular masses of the mature FedB and FedC are 86,001 and 23,418 Da, respectively. The fedF gene was also PCR cloned and sequenced from a Finnish E. coli O141 isolate (data not shown) and found to have 99.6% identity with the fedF derived from pIH120. In addition to the previously reported transcription terminator, located downstream of fedA (5), an inverted repeat (⌬G of Ϫ17.3 kcal mol Ϫ1 ) for th...

Evolution of the Chaperone/Usher Assembly Pathway: Fimbrial Classification Goes Greek

“…In the case of F18 fibrillae, the major subunit FedA is assembled into a helix containing two subunits per turn (123). Minor fimbrial subunits have been implicated in the adherence of F18 fimbriae and AF/R1 fimbriae (48,148,307). However, in the case of F4 and F5 fimbriae, minor subunits are dispensable for adherence (22,303,304), and the major fimbrial subunits (FaeG and FanC) have been implicated in determining the binding activity of their respective adhesins (21,156,250).…”

“…7). The -fimbriae (FUP cluster 6) (367) comprise usher proteins required for the assembly of the F4 fimbrial antigen (K88 fimbriae) (FaeD) (20,22,224,245,315,316,338), the F5 fimbrial antigen (K99 fimbriae) (FanD) (1, 20, 149, 151, 247, [275][276][277]303), and the F18 fimbrial antigen (F107 fimbriae) (FedB) (147,148,307) found in porcine and bovine isolates of enterotoxigenic E. coli (51,362). Furthermore, this group includes adherence factor/rabbit 1 (AF/R1) fimbriae (AfrB) (48), an uncharacterized operon of E. coli (CshB), the locus for diffuse adherence (LdaD) of atypical enteropathogenic E. coli (294), plasmid-encoded fimbriae (PefC) of S. enterica serotype Typhimurium (96,218), and an intestinal colonization factor of rabbit enteropathogenic E. coli (RalD) (2).…”

Receptor Binding Domain ofEscherichia coliF18 Fimbrial Adhesin FedF Can Be both Efficiently Secreted and Surface Displayed in a Functional Form inLactococcus lactis

“…The functionality of this approach has been recently demonstrated with model systems (9,10,25). Regarding the adhesin of F18 fimbriae, we have shown that E. coli strains carrying this fimbrial type adhere to porcine intestinal epithelial cells via the FedF protein (42). Furthermore, we have recently mapped the receptor binding region of FedF, to be used as a putative surface antigen in lactic acid bacteria (LAB), between amino acid (aa) residues 60 and 109 of FedF (43).…”

Cloning and Expression of Genes Encoding F107-C and K88-1NT Fimbrial Proteins of Enterotoxigenic Escherichia coli from Piglets

“…Two additional genes from the fed gene cluster, fedE and fedF, have been described as essential for fimbrial adhesion and fimbrial length [11]. However, until now it can not to evaluate F18 adhesive function in regard to either of the two gene products [12].…”