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CfaE tip mutations in enterotoxigenic Escherichia coli CFA/I fimbriae define critical human intestinal binding sites - PubMed

CfaE tip mutations in enterotoxigenic Escherichia coli CFA/I fimbriae define critical human intestinal binding sites

K K Baker et al. Cell Microbiol. 2009 May.

Abstract

Enterotoxigenic Escherichia coli (ETEC) use colonization factors to attach to the human intestinal mucosa, followed by enterotoxin expression that induces net secretion and diarrhoeal illness. ETEC strain H10407 expresses CFA/I fimbriae, which are composed of multiple CfaB structural subunits and a CfaE tip subunit. Currently, the contribution of these individual fimbrial subunits in intestinal binding remains incompletely defined. To identify the role of CfaE in attachment in the native ETEC background, an R181A single-amino-acid substitution was introduced by recombination into the H10407 genome. The substitution of R181A eliminated haemagglutination and binding of intestinal mucosa biopsies in in vitro organ culture assays, without loss of CFA/I fimbriae expression. Wild-type in trans plasmid-expressed cfaE restored the binding phenotype. In contrast, in trans expression of cfaE containing amino acid 181 substitutions with similar amino acids, lysine, methionine and glutamine did not restore the binding phenotype, indicating that the loss of the binding phenotype was due to localized areas of epitope disruption. R181 appears to have an irreplaceable role in the formation of a receptor-binding feature on CFA/I fimbriae. The results specifically indicate that the CfaE tip protein is a required binding factor in CFA/I-mediated ETEC colonization, making it a potentially important vaccine antigen.

© 2009 Blackwell Publishing Ltd.

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Figures

Fig. 1
Fig. 1

Genetic outline of the CFA/I operon. H10407 contains the wild-type operon. H10407Kan possesses an aph insertion at base pair 5228 within the cfaE gene of the CFA/I operon (NCBI M55661). KB101 contains base pair substitutions of AGA to GCA (base pairs 5323–5325). PCR and RT-PCR analyses were performed with primers CFAE1 and CFAE2 flanking the R181 region and the KAN-REV primer that anneals in aph.

Fig. 2
Fig. 2

Genotypic confirmation of H10407 and derivatives by PCR. PCR was conducted with primers CFAE1 and CFAE2 using the following bacterial strains as templates: Lane 1, Fermentas 1 kb Generuler; lane 2, H10407; lane 3, H10407Kan and lane 4, KB101.

Fig. 3
Fig. 3

Western blot analysis of CfaB expression. Whole-cell lysates of ETEC strains were probed with anti-CFA/I polyclonal antibody. Lanes are as follows: 1, molecular weight markers; 2, H10407; 3, H10407P; 4, H10407Kan; 5, KB101; 6, H10407Kan(pcfaEgent); KB101(pcfaE). The asterisk (*) indicates CfaB major structural protein band at 15 kDa.

Fig. 4
Fig. 4

RT-PCR analysis of cfaE transcription in H10407 and derivative strains. Amplification of cDNA products was performed with primer pairs CFAE1 and CFAE2 (A), or CFAE1 and Kan-rev (B). Lanes are as follows: 1, Fermentas 1 kb Generuler marker; 2, control lacking reverse transcriptase enzyme; 3, control lacking RNA; 4, H10407; 5, H10407P; 6, H10407Kan; 7, KB101; 8, H10407Kan(pcfaEgent) and 9, KB101(pcfaE).

Fig. 5
Fig. 5

Expression of CFA/I fimbriae assessed by transmission electron microscopy. ETEC strains were stained with 2% ammonium molybdate, and photographed at 30× magnification. A. H10407. B. H10407Kan. C. KB101. D. H10407Kan(pcfaEgent).

Fig. 6
Fig. 6

Confirmation of CFA/I fimbrial expression by immunogold electron microscopy. WT and mutant ETEC derivatives were stained with anti-CFA/I antibody. A. H10407 at 30× magnification. B. H10407 at 50× magnification. C. KB101 at 25× magnification. D. KB101 80× magnification.

Fig. 7
Fig. 7

MRHA testing ETEC strain binding of human erythrocytes. A. H10407. B. H10407P. C. H10407Kan. D. KB101. E. H10407Kan(pcfaEgent). F. KB101(pcfaE). All strains were normalized to an OD600 of 1.0. Drops were made by combining 50 μl each of bacteria, 1% D-Mannose and 3% human Type A red blood cells and mixing with gentle stirring at room temperature.

Fig. 8
Fig. 8

IVOC of ETEC strains with human small intestine. A. H10407 adhering to mucosal surface (bar = 10 μm); inset shows bacterial detail (bar = 1 μm). B. Surface detail of mucosa incubated with H10407P showing no evidence of bacterial adhesion (bar = 10 μm). C. Mucosal surface without adhering bacteria in KB101 IVOC (bar = 5 μm); inset shows single adhering bacillus (bar = 1 μm). D. KB101(pcfaE) demonstrating mucosal bacterial adhesion (bar = 5 μm); inset shows detail of adhering bacteria (bar = 1 μm).

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