title: Structure-function analysis of the enteroaggregative Escherichia coli plasmid-encoded toxin autotransporter using scanning linker mutagenesis.
abstract: The plasmid-encoded toxin (Pet) from enteroaggregative Escherichia coli is a cytopathic serine protease, which is prototypical of a large family of bacterial autotransporter toxins. To further elucidate the structure-function relationships of this toxin, we employed transposon-based scanning linker mutagenesis. A subset of insertions throughout the Pet mature toxin (passenger) domain reduced secretion to the extracellular space. Many of these mutants were undetectable, but secretion of a subset of mutants with insertions in the N-terminal half of the toxin could be restored to wild type secretion levels if cultured in the presence of 0.1% Triton X-100. Secretion of two mutants with insertions at the extreme C terminus was partially restored when co-expressed with a minimal clone of EspP, a related autotransporter protein. Several well secreted mutants with insertions in the N-terminal third of the molecule reduced protease activity over 20-fold, suggesting that the protease domain is located within this N-terminal region of Pet. We have also identified two insertional mutants in the middle of the passenger domain that were proteolytic but no longer cytopathic; these mutants displayed decreased binding and internalization upon incubation with HEp-2 cells. Our data suggest the existence of separate functional domains mediating Pet proteolysis, secretion, and cell interaction.
authors.affiliation: Center for Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
authors.forename: Pinaki R
authors.initials: PR
authors.lastname: Dutta
authors.forename: Bao Quan
authors.initials: BQ
authors.lastname: Sui
authors.forename: James P
authors.initials: JP
authors.lastname: Nataro
mesh_terms: D001427:Bacterial Toxins / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity*; D001483:Base Sequence; D002460:Cell Line; D004269:DNA, Bacterial / Q000235:genetics; D004768:Enterotoxins / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity*; D004926:Escherichia coli / Q000235:genetics* / Q000472:pathogenicity* / Q000502:physiology; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000633:toxicity*; D006801:Humans; D016254:Mutagenesis, Insertional; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D017434:Protein Structure, Tertiary; D012697:Serine Endopeptidases; D013329:Structure-Activity Relationship; D014774:Virulence / Q000235:genetics / Q000502:physiology
chemical_list: D001427:Bacterial Toxins; D004269:DNA, Bacterial; D004768:Enterotoxins; D029968:Escherichia coli Proteins; C027538:heat-labile enterotoxin, E coli; C530786:Pet protein, E coli; D012697:Serine Endopeptidases