Interaction of an autotransporter passenger domain with BamA during its translocation across the bacterial outer membrane.
Autotransporters are a superfamily of virulence factors produced by Gram-negative bacteria consisting of a large N-terminal extracellular domain ("passenger domain") and a C-terminal beta barrel domain ("beta domain"). The mechanism by which the passenger domain is translocated across the outer membrane (OM) is unknown. Here we show that the insertion of a small linker into the passenger domain of the Escherichia coli O157:H7 autotransporter EspP effectively creates a translocation intermediate by transiently stalling translocation near the site of the insertion. Using a site-specific photocrosslinking approach, we found that residues adjacent to the stall point interact with BamA, a component of a heterooligomeric complex (Bam complex) that catalyzes OM protein assembly, and that residues closer to the EspP N terminus interact with the periplasmic chaperones SurA and Skp. The EspP-BamA interaction was short-lived and could be detected only when passenger domain translocation was stalled. These results support a model in which molecular chaperones prevent misfolding of the passenger domain before its secretion and the Bam complex catalyzes both the integration of the beta domain into the OM and the translocation of the passenger domain across the OM in a C- to N-terminal direction.
Genetics and Biochemistry Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Raffaele
R
Ieva
Harris D
HD
Bernstein
D001425:Bacterial Outer Membrane Proteins / Q000378:metabolism*; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D019453:Escherichia coli O157 / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D008956:Models, Chemical; D018832:Molecular Chaperones / Q000378:metabolism; D019696:Peptidylprolyl Isomerase / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D012697:Serine Endopeptidases / Q000235:genetics / Q000378:metabolism*; D037521:Virulence Factors / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; C503914:BamA protein, E coli; D002352:Carrier Proteins; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D018832:Molecular Chaperones; D037521:Virulence Factors; C072721:Skp protein, E coli; C107189:EspP protein, E coli; D012697:Serine Endopeptidases; C490900:SurA protein, E coli; D019696:Peptidylprolyl Isomerase