Structural and functional properties of chimeric EspA-FliCi filaments of EPEC.
Enteropathogenic Escherichia coli utilise a filamentous type III secretion system to translocate effector proteins into host gut epithelial cells. The primary constituent of the extracellular component of the filamentous type III secretion system is EspA. This forms a long flexible helical conduit between the bacterium and host and has a structure almost identical to that of the flagella filament. We have inserted the D3 domain of FliCi (from Salmonella typhimurium) into the outer domain of EspA and have studied the structure and function of modified filaments when expressed in an enteropathogenic E. coli espA mutant. We found that the chimeric protein EspA-FliCi filaments were biologically active as they supported protein secretion and translocation [assessed by their ability to trigger actin polymerisation beneath adherent bacteria (fluorescent actin staining test)]. The expressed filaments were recognised by both EspA and FliCi antisera. Visualisation and analysis of the chimeric filaments by electron microscopy after negative staining showed that, remarkably, EspA filaments are able to tolerate a large protein insertion without a significant effect on their helical architecture.
Crepin
Valerie F
VF
Division of Cell and Molecular Biology, Flowers Building, Imperial College London, London SW7 2AZ, UK.
Martinez
Eric
E
Shaw
Robert K
RK
Frankel
Gad
G
Daniell
Sarah J
SJ
D000595:Amino Acid Sequence; D054308:Enteropathogenic Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005408:Flagellin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000235:genetics / Q000378:metabolism
D029968:Escherichia coli Proteins; C102051:EspA protein, E coli; D011993:Recombinant Fusion Proteins; D005408:Flagellin