B-297. Oligomerization of the Type II Secretion Protein EpsE as a Means of Stimulating Its ATPase Activity

M. Patrick¹, J. L. Camberg², M. Sandkvist¹;
¹Univ. of Michigan, Ann Arbor, MI, ²NIH, Bethesda, MD.

The diarrheal disease cholera propagates due to the extracellular secretion of cholera toxin from the gram negative bacterium, Vibrio cholerae. Secretion of the toxin and other degradative enzymes is mediated by the type II secretion (T2S) system; a pathway conserved in many pathogenic species. In V. cholerae at least 13 proteins, EpsC-EpsN and PilD, assemble into a multiprotein complex spanning the inner and outer membranes. The energy to sustain a functional T2S system is thought to be provided by the cytoplasmic ATPase, EpsE. EpsE primarily purifies as a monomer with low ATPase activity; however, the presence of acidic phospholipids and the T2S inner membrane protein EpsL increases its ATPase activity significantly. This increase correlates with increased levels of EpsE oligomers, suggesting that the oligomeric form may be the active form of EpsE. Although EpsE purifies as a monomer and crystallizes as a filament, its active form has been modeled as a hexameric ring based on structural homology with other traffic ATPases. We hypothesize that EpsE oligomerizes in the presence of EpsL and acidic phospholipids, which consequently promotes the stimulation of EpsE ATPase activity. To test this hypothesis, residues implicated in oligomerization of EpsE were mutated in order to disrupt putative subunit-subunit interactions. When tested in vivo, these mutant proteins failed to complement an epsE deficient strain of V. cholerae, and exhibited a dominant negative effect on the secretion in wild type V. cholerae. In vitro experiments to test the ATPase activity of purified proteins in the presence of EpsL and acidic phospholipids showed reduced activity levels of the mutant proteins when compared to wild type EpsE. The basal, unstimulated activities of both mutant and wild type proteins were, however, comparable. These results suggest that the mutations caused a defect in stimulating EpsE ATPase activity and not necessarily in the ATP binding or hydrolysis, per se. Although confirmation of these data awaits further investigation, these results strengthen the model that EpsE functions as an oligomer.