Molecular basis of antigenic polymorphism of EspA filaments: development of a peptide display technology.
Like many Gram-negative pathogens, enteropathogenic (EPEC) and enterohaemorrhagic Escherichia coli (EHEC) use a macromolecular type III secretion system (TTSS) to inject effector proteins into eukaryotic host cells. The membrane-associated needle complex (NC) of the TTSS, which shows broad similarity to the flagellar basal body, is conserved amongst bacterial pathogens. However, the extracellular part of the TTSS of EPEC and EHEC is unique, in that it has a hollow, approximately 12 nm in diameter, filamentous extension to the NC. EspA filaments are homo-polymers made of the translocator protein EspA. The three-dimensional structure of EspA filaments is comparable to that of flagella; the helical symmetry and packing of the subunits forming both filamentous structures are very similar. Like flagella, EspA filaments show antigenic polymorphism as EspA from different EPEC and EHEC clones show no immunological cross-reactivity. In this study, we determined the molecular basis of the antigenic polymorphism of EspA filaments and identified a surface-exposed hypervariable domain that contains the immunodominant EspA epitope. By exchanging the hypervariable domains of EspA(EPEC) and EspA(EHEC) we swapped the antigenic specificity of the EspA filaments. As for the flagellin D3 domain, which is known to tolerate insertions of natural and artificial amino acid sequences, we have inserted short peptides into the surface-exposed, hypervariable domain of EspA. We demonstrated that the inserted peptides are presented on the surface of the recombinant EspA filaments forming a new immunodominant epitope. Accordingly, EspA filaments have a potential to be developed into a novel epitope display system.
Crepin
Valérie F
VF
Centre for Molecular Microbiology and Infection, Flowers Building, Imperial College London, London SW7 2AZ, UK.
Shaw
Robert
R
Knutton
Stuart
S
Frankel
Gad
G
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D057927:Hydrophobic and Hydrophilic Interactions; D008969:Molecular Sequence Data; D019151:Peptide Library; D011110:Polymorphism, Genetic / Q000235:genetics*; D016415:Sequence Alignment
D000199:Actins; D000942:Antigens, Bacterial; D000939:Epitopes; D029968:Escherichia coli Proteins; C102051:EspA protein, E coli; D019151:Peptide Library