matB , a Common Fimbrillin Gene of Escherichia coli , Expressed in a Genetically Conserved, Virulent Clonal Group

Abstract

A novel fimbrial type in Escherichia coli was identified and characterized. The expression of the fimbria was associated with the O18acK1H7 clonal group of E. coli, which cause newborn meningitis and septicemia when grown at low temperature; hence, it was named the Mat (meningitis associated and temperature regulated) fimbria. The fimbriae were purified from a fimA::cat sfaA::Gm fliC::St derivative of the O18K1H7 isolate E. coli IHE 3034. The purified Mat fimbrillin had an apparent molecular mass of 18 kDa and did not serologically cross-react with the type 1 or S fimbria of the same strain. The matB gene encoding the major fimbrillin was cloned from the genomic DNA of the fimA::cat sfaA::Gm fliC::St derivative of IHE 3034. The predicted MatB sequence was of 195 amino acids, contained a signal sequence of 22 residues, and did not show significant homology to any of the previously characterized fimbrial proteins. The DNA sequence of matB was 97.8% identical to a region from nucleotides 17882 to 18469 in the 6-to 8-min region of the E. coli K-12 chromosome, reported to encode a hypothetical protein. The 7-kb DNA fragment containing matB of IHE 3034 was found by restriction mapping and partial DNA sequencing to be highly similar to the corresponding region in the K-12 chromosome. Trans complementation of the matB::cat mutation in the IHE 3034 chromosome showed that matB in combination with matA or matC restored surface expression of the Mat fimbria. A total of 27 isolates representing K-12 strains and the major pathogroups of E. coli were analyzed for the presence of a matB homolog as well as for expression of the Mat fimbria. A conserved matB homolog was found in 25 isolates; however, expression of the Mat fimbriae was detected only in the O18acK1H7 isolates. Expression of the Mat fimbria was temperature regulated, with no or a very small amount of fimbriae or intracellular MatB fimbrillin being detected in cells cultivated at 37 o C. Reverse transcriptase PCR and complementation assays with mat genes controlled by the inducible trc promoter indicated that regulation of Mat fimbria expression involved both transcriptional and posttranscriptional events. Numerous proteinaceous adhesins have been detected inEscherichia coli (for recent reviews, see references 20 and 27). These adhesins occur in the form of fimbrial filaments or are nonfimbrial proteins of the outer membrane. The adhesins recognize different receptor molecules on the mammalian epithelia or extracellular matrices and function to enable colonization of E. coli at specific ecological niches. Many of the adhesins are associated with E. coli isolates from specific disease manifestations and contribute to the establishment of the infections. Examples of such disease-associated adhesins include the P fimbria of uropathogenic E. coli (UPEC) (55) and the various adhesin types detected in E. coli pathogroups causing diarrheal diseases (reviewed in references 13 and 36). Some adhesin genes, such as those encoding the mannosidebinding type 1 fimbriae (26) and th...

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

“…The presence of host cells was not required for ECP production as initially thought, eliminating the hypothesis that eukaryotic cells or a soluble product were triggering signals. In contrast to meningitis-associated E. coli (33), EHEC produced ECP after growth in DMEM at 26°C or 37°C, but not in LB; suggesting that subtle differences in the mechanisms regulating ECP production may exist between different strains. Production of ECP at temperatures Ͻ37°C might have important implications during the life of the organism outside their bovine or human hosts, allowing for their persistence in the environment and contamination of produce.…”

“…Expression of bacterial pili is under the influence of environmental cues and, sometimes, host factors (35). Previously, it was shown that meningitis-associated E. coli strains, but not any other E. coli pathogroups, were able to assemble the EcpA protein (formerly YagZ) into pili named ''Mat'' (meningitisassociated and temperature-regulated pilus) only after growth at 20°C in LB broth (33). EHEC is indeed able to assemble the EcpA protein into pili upon infection of cultured epithelial cells at host temperature, a phenotype that is biologically significant from the host-pathogen interaction standpoint.…”

“…The major component of these pili was resolved as a 21-kDa protein (Fig. 2B, lane 1) whose size and N terminus amino acid sequence matched that of the predicted protein encoded by the yagZ gene found in the genome of EHEC O157:H7 (6,8), E. coli K-12 (5), uropathogenic E. coli (7), and meningitis-associated E. coli (33). In light of the apparent wide distribution of yagZ among the E. coli, we propose the generic name ''E.…”

“…coli common pilus'' (ECP) for the pilus composed by the subunit protein product of this gene, herein proposed to be renamed ''ecpA.'' Sequence analysis of the predicted EcpA subunit revealed that 60% of the protein is hydrophobic (33) and its C terminus does not contain the typical two-cysteine residues present in many pili types (34). Rabbit polyclonal antibodies produced against purified ECP, specifically detected the 21-kDa protein by immunoblotting (IB) (Fig.…”

“…Immuno-EM was performed with anti-EHEC ECP antibodies and anti-rabbit IgG conjugated to 10-or 30-nm gold particles as described (38). Initial studies used rabbit anti-MatB antibodies (kindly provided by Timo K. Korhonen, University of Helsinki, Helsinki, Finland) (33). Glass coverslips containing fixed mammalian cells with adhering bacteria were prepared for SEM as described (38) and visualized by using a Hitachi S-4500 scanning electron microscope (Hitachi, Tokyo, Japan).…”