Display of passenger proteins on the surface of Escherichia coli K-12 by the enterohemorrhagic E. coli intimin EaeA.
Intimins are members of a family of bacterial adhesins from pathogenic Escherichia coli which specifically interact with diverse eukaryotic cell surface receptors. The EaeA intimin from enterohemorrhagic E. coli O157:H7 contains an N-terminal transporter domain, which resides in the bacterial outer membrane and promotes the translocation of four C-terminally attached passenger domains across the bacterial cell envelope. We investigated whether truncated EaeA intimin lacking two carboxy-terminal domains could be used as a translocator for heterologous passenger proteins. We found that a variant of the trypsin inhibitor Ecballium elaterium trypsin inhibitor II (EETI-II), interleukin 4, and the Bence-Jones protein REI(v) were displayed on the surface of E. coli K-12 via fusion to truncated intimin. Fusion protein net accumulation in the outer membrane could be regulated over a broad range by varying the cellular amount of suppressor tRNA that is necessary for translational readthrough at an amber codon residing within the truncated eaeA gene. Intimin-mediated adhesion of the bacterial cells to eukaryotic target cells could be mimicked by surface display of a short fibrinogen receptor binding peptide containing an arginine-glycine-aspartic acid sequence motif, which promoted binding of E. coli K-12 to human platelets. Cells displaying a particular epitope sequence fused to truncated intimin could be enriched 200,000-fold by immunofluorescence staining and fluorescence-activated cell sorting in three sorting rounds. These results demonstrate that truncated intimin can be used as an anchor protein that mediates the translocation of various passenger proteins through the cytoplasmic and outer membranes of E. coli and their exposure on the cell surface. Intimin display may prove a useful tool for future protein translocation studies with interesting biological and biotechnological ramifications.
Wentzel
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Abteilung für Molekulare Genetik und Präparative Molekularbiologie, Institut für Mikrobiologie und Genetik, Georg-August-Universität Göttingen, D-37077 Göttingen, Germany.
Christmann
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Adams
T
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Kolmar
H
H
D018829:Adhesins, Bacterial / Q000096:biosynthesis* / Q000235:genetics; D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000276:immunology / Q000302:isolation & purification; D001422:Bacterial Adhesion; D002352:Carrier Proteins / Q000096:biosynthesis* / Q000235:genetics; D000939:Epitopes; D004926:Escherichia coli / Q000254:growth & development / Q000302:isolation & purification; D019453:Escherichia coli O157 / Q000254:growth & development / Q000302:isolation & purification; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D029082:Sendai virus / Q000276:immunology
D018829:Adhesins, Bacterial; D000956:Antigens, Viral; D002352:Carrier Proteins; D000939:Epitopes; D029968:Escherichia coli Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; C073819:eaeA protein, E coli