Novel type of fimbriae encoded by the large plasmid of sorbitol-fermenting enterohemorrhagic Escherichia coli O157:H(-)

Abstract

Sorbitol-fermenting (SF) enterohemorrhagic Escherichia coli (EHEC) O157:H(-) have emerged as important causes of diarrheal diseases and the hemolytic-uremic syndrome in Germany. In this study, we characterized a 32-kb fragment of the plasmid of SF EHEC O157:H(-), pSFO157, which differs markedly from plasmid pO157 of classical non-sorbitol-fermenting EHEC O157:H7. We found a cluster of six genes, termed sfpA, sfpH, sfpC, sfpD, sfpJ, and sfpG, which mediate mannose-resistant hemagglutination and the expression of fimbriae. sfp genes are similar to the pap genes, encoding P-fimbriae of uropathogenic E. coli, but the sfp cluster lacks homologues of genes encoding subunits of a tip fibrillum as well as regulatory genes. The major pilin, SfpA, despite its similarity to PapA, does not cluster together with known PapA alleles in a phylogenetic tree but is structurally related to the PmpA pilin of Proteus mirabilis. The putative adhesin gene sfpG, responsible for the hemagglutination phenotype, shows significant homology neither to papG nor to other known sequences. Sfp fimbriae are 3 to 5 nm in diameter, in contrast to P-fimbriae, which are 7 nm in diameter. PCR analyses showed that the sfp gene cluster is a characteristic of SF EHEC O157:H(-) strains and is not present in other EHEC isolates, diarrheagenic E. coli, or other Enterobacteriaceae. The sfp gene cluster is flanked by two blocks of insertion sequences and an origin of plasmid replication, indicating that horizontal gene transfer may have contributed to the presence of Sfp fimbriae in SF EHEC O157:H(-).

FIG. 1

Genetic organization of the sfp fimbrial gene cluster and comparison with the pap cluster. Functions of Pap proteins (22) are shown below the map. Shaded boxes indicate components only present in the pap cluster. The black box depicts a DNA region with high homology to IS2. The double arrows indicate regions amplified by sfpA PCR (A) and sfpDG PCR (DG).

FIG. 2

Conserved structural motifs within Sfp subunits. Putative amino acid sequences of the C-terminal β-zipper-forming motif as well as the second site of interaction between pilins and the periplasmic chaperone are compared to the respective motifs of PapA and PapG (17). The most conserved residues are indicated by shaded boxes, and the asterisks mark the alternating hydrophobic amino acids in the C domain (open asterisks stand for residues conserved in all pilins with the exception of SfpJ).

FIG. 3

Phylogenetic relationship between SfpA and different PapA alleles. Included are 11 PapA variants of E. coli (F7–1, F7–2, F8, F9, F10, F11, F12, F13, F14, F15, and F16), the Pap-related SmfA, MrpA, and PmpA pilins of S. marcescens and P. mirabilis, and the E. coli type I (FimA) and S-fimbriae (SfaA) major subunits. The unrooted tree was based on (predicted) mature peptides and was constructed using the neighbor-joining method. The presumed evolutionary distance between any two members of the tree equals the sum of the lengths of the branches connecting them.

FIG. 4

Sequence comparison between SfpG and PapG_F13 (36). The alignment was produced using CLUSTAL W. Identical amino acid residues are depicted by boxes. The predicted signal peptide as well as the sites responsible for the interaction between PapG and the periplasmic chaperone (second site and C domain) are indicated by the brackets. Conserved pairs of cysteine residues in the C-terminal protein domain are marked by asterisks.

FIG. 5

(A) Genetic organization of the sfp gene cluster within plasmid pSFO157. White boxes indicate open reading frames, and shaded boxes depict regions with homology to IS elements. Putative origins of replication (RepFII and RepFIB) are indicated below the map. The brackets in the lower row show the localization of plasmid fragments cloned in this study. The double arrow labeled “wprom” indicates the PCR amplicon used for the sequencing of the connection between fragment pSFO157-E14 and pSFO157-E11. The arrow labeled “G-Apa” depicts the location of the PCR product used for the cloning of sfpG. Above the gene map, nearly identical sequences of plasmid pO157 of NSF EHEC O157:H7 strain EDL933 are depicted by the bold line together with their sequence positions (GenBank AF074613). The ruler depicts map positions of pSFO157 in kilobase pairs. (B) Map of the recombinant plasmid pSFO157-ESn8::sfpG^Apa. The G-Apa PCR amplicon was inserted into the ApaI restriction site of the sfpG mutant plasmid pSFO157-ESn8. Plasmid parts derived from the vector pBluescript II KS are depicted as a bold black line together with relevant restriction sites (A, ApaI; E, EcoRI; K, KpnI; Sm, SmaI; Sn, SnaBI; St, SstI). Arrows indicate the transcription direction of the ORFs. Nonfunctional ORFs are given in parentheses.

FIG. 6

Coomassie blue-stained SDS-PAGE (A) and immunoblot analysis using anti-SfpA antiserum (B) of thermoeluted fimbrial extracts taken from recombinant E. coli strains HB101/pSFO157-E11 (lane 1), HB101/pBluescript II KS (vector control) (lane 2), HB101/pSFO157-ESn8 (sfpG mutant) (lane 3), and pSFO157-ESn8::sfpG^Apa (lane 4). Positions and sizes of marker proteins (in kilodaltons) are given on the left. The arrows indicate the SfpA protein as identified by amino acid sequencing. Titers of mannose-resistant hemagglutination (HA) of the strains are given below the immunoblot. The bands strongly reacting with anti-SfpA antiserum above and below the SfpA protein and not visible in Coomassie blue-stained SDS-PAGE are suspected to represent nonproteinaceous components of the bacterial cell envelope present in the SfpA preparation used for immunization. These bands were not detected using rabbit normal serum, nor were they visible in silver-stained gels.

FIG. 7

Expression of fimbriae in recombinant E. coli K-12 strains harboring the sfp gene cluster shown by electron microscopy. (A) Strain HB101/pSFO157-E11; (B) sfpG mutant strain HB101/pSFO157-ESn8; (C) complemented mutant HB101/pSFO157-ESn8::sfpG^Apa. The bar represents 200 nm.