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 Esch erichia coli which specifically interact with diverse eukaryotic cell surfa ce receptors. The EaeA intimin from enterohemorrhagic E. coli O157:H7 conta ins an N-terminal transporter domain, which resides in the bacterial outer membrane and promotes the translocation of four C-terminally attached passe nger domains across the bacterial cell envelope. We investigated whether tr uncated 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, were displayed on the su rface of E. coli K-12 via fusion to truncated intimin. Fusion protein net a ccumulation in the outer membrane could be regulated over a broad range by varying the cellular amount of suppressor tRNA that is necessary for transl ational readthrough at an amber codon residing within the truncated eaeA ge ne. Intimin-mediated adhesion of the bacterial cells to eukaryotic target c ells could be mimicked by surface display of a short fibrinogen receptor bi nding 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 cel l sorting in three sorting rounds. These results demonstrate that truncated intimin can be used as an anchor protein that mediates the translocation o f 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 bio logical and biotechnological ramifications.