B-284. Roles for Major Periplasmic Chaperones in the Biogenesis of Autotransporter Proteins from the SPATE Family

F. Ruiz--Perez, J. P. Nataro;
Univ. of Maryland, Baltimore, MD.

Background. The autotransporter secretion system (AT) is the most common mechanism of outer membrane (OM) translocation. The mechanism comprises entry to the periplasm via the Sec apparatus, followed by formation of an outer membrane β-barrel, which hypothetically allows passage of the N-terminal passenger domain to the extracellular milieu. The roles of accessory proteins in AT translocation are not currently known. Here we studied the role of the major periplasmic chaperones (Skp, SurA, DegP, FkpA, PpiA and PpiD) and the essential outer membrane YaeT protein in the biogenesis of the EspP protein from Enterohemorrhagic E. coli, a prototype autotransporter protein. Methods. We employed the yeast two hybrid (Y2H) system, overlay protocols, and the use of mutant strains to reveal protein-protein interactions. Results. Y2H and/or overlay experiments suggested interactions between the EspP protein and the periplasmic SurA, Skp, DegP and the outer membrane YaeT protein. These proteins each interacted with the EspP beta domain. Surprisingly, we also found evidence for interaction of the SurA chaperone protein with the EspP passenger domain. Secretion of EspP was impaired in SurA and Skp mutants. Moreover, secretion of SPATE members was drastically reduced in a DegP mutant. Analysis of the outer membrane proteins from all mutant strains confirmed the presence of the AT translocation domain at the same levels as the wild type; mutants yielded reduced levels of the EspP periplasmic intermediate, suggesting degradation of the passenger domain. Proscan analysis identified 12 putative SurA/DegP motifs (“aro-x-aro”) on the EspP molecule. 4 potential SurA/DegP motifs were highly conserved throughout the SPATE family. Combined site directed mutagenesis on three conserved putative motifs showed drastically reduction of the EspP secretion, though individual mutations did not produce this phenotype. Conclusion: Our data suggest the direct involvement of SurA and DegP chaperones in AT biogenesis. Contributions of these chaperones was most likely during translocation of the passenger domain, rather than on insertion of the beta domain to the OM.