Outer Membrane Secretion Efficiency of Autotransporter Virulence Proteins Correlates with Passenger Domain Folding Properties

Abstract

Autotransporters (ATs) are the largest class of extracellular virulence proteins secreted from Gram-negative bacteria. Each AT is synthesized as a tripartite pre-protein containing an N-terminal signal sequence that directs secretion across the inner membrane, a central “passenger” domain that becomes the mature extracellular virulence protein, and a C-terminal outer membrane (OM) porin domain that is essential for OM transport. AT passenger domains have highly diverse sequences, lengths, and functions, but almost all are predicted to contain β-helical structure. It was originally proposed that an AT protein autonomously catalyzes transport of its own passenger domain across the OM, but recent results have cast doubt on this model. Moreover, in the absence of a significant quantity of ATP or proton gradient across the OM, the driving force for efficient OM secretion remains unclear. Here we demonstrate a direct correlation between localized regions of AT passenger domain stability (ΔGunfolding) and OM secretion efficiency. Destabilizing the C-terminus of a passenger domain β-helix significantly reduced OM secretion efficiency. In contrast, destabilizing more N-terminal portions of the passenger domain produced a linearly correlated increase in OM secretion efficiency. Thus, C-terminal passenger domain stability facilitates OM secretion, whereas N-terminal stability hinders it. The contributions of regional passenger stability to OM secretion efficiency demonstrate a crucial role for the passenger domain itself in directing its secretion across the OM. These results indicate that the folding properties of distinct regions of the passenger domain provide a directed molecular driving force, for use as a generalized transporter device. The regionalized distribution of AT passenger domain stability provides a unique solution for the directed transport of macromolecules across biological membranes, defining a new category of ATP-independent Brownian motor.