Multiple Driving Forces Required for Efficient Secretion of Autotransporter Virulence Proteins

Igor Drobnak,Esther Braselmann,Patricia L Clark

doi:10.1074/jbc.m114.629170

Igor Drobnak, Esther Braselmann + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m114.629170

Copy DOI

Abstract

Autotransporter (AT) proteins are a broad class of virulence proteins from Gram-negative bacterial pathogens that require their own C-terminal transmembrane domain to translocate their N-terminal passenger across the bacterial outer membrane (OM). But given the unavailability of ATP or a proton gradient across the OM, it is unknown what energy source(s) drives this process. Here we used a combination of computational and experimental approaches to quantitatively compare proposed AT OM translocation mechanisms. We show directly for the first time that when translocation was blocked an AT passenger remained unfolded in the periplasm. We demonstrate that AT secretion is a kinetically controlled, non-equilibrium process coupled to folding of the passenger and propose a model connecting passenger conformation to secretion kinetics. These results reconcile seemingly contradictory reports regarding the importance of passenger folding as a driving force for OM translocation but also reveal that another energy source is required to initiate translocation.

Highlights

Many virulence proteins are secreted using the autotransporter system
We found that pertactin outer membrane (OM) translocation is tightly coordinated with folding: no folding occurred in a wild type passenger that is reversibly stalled in the periplasm, and a mutant deficient in passenger folding exhibited a marked decrease in OM translocation efficiency
The first requirement of secretion driven by passenger folding is the inability of the folded passenger to re-enter the periplasm, which is satisfied because the dimensions of the folded passenger exceed the pore diameter of the fully folded ␤-barrel [22, 29] and the OM is generally impermeable to proteins

Summary

Background

Many virulence proteins are secreted using the autotransporter system. Results: Autotransporter proteins do not fold until secreted and are secreted poorly in the absence of folding. We demonstrate that AT secretion is a kinetically controlled, non-equilibrium process coupled to folding of the passenger and propose a model connecting passenger conformation to secretion kinetics These results reconcile seemingly contradictory reports regarding the importance of passenger folding as a driving force for OM translocation and reveal that another energy source is required to initiate translocation. The challenge of AT translocation across the OM is that beyond the inner membrane no such obvious external energy source is available to couple to and drive AT secretion This means that during OM translocation the AT protein must progressively move from a less stable (high energy) to a more stable (low energy) state throughout this process; if it falls into an off-pathway low energy trap, there is no ATP-driven mechanism available to restore efficient secretion. From an entropic perspective, concentrating all AT mole-

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION