Stepwise Folding of an Autotransporter Passenger Domain Is Not Essential for Its Secretion

Background: It has been proposed that stepwise folding of the extracellular (passenger) domains of bacterial autotransporter proteins drives their secretion. Results: Mutations in N-terminal or medial segments of a passenger domain that severely impair folding only moderately reduce secretion efficiency. Conclusion: Stepwise folding only partially accounts for the energetics of autotransporter secretion. Significance: Autotransporter secretion may involve the use of a novel energy source. Autotransporters are a superfamily of virulence proteins produced by Gram-negative bacteria. They consist of an N-terminal -helical domain (passenger domain) that is secreted into the extracellular space and a C-terminal -barrel domain (-domain) that anchors the protein to the outer membrane. Because the periplasm lacks ATP, vectorial folding of the passenger domain in a C-to-N-terminal direction has been proposed to drive the secretion reaction. Consistent with this hypothesis, mutations that disrupt the folding of the C terminus of the passenger domain of the Escherichia coli O157:H7 autotransporter EspP have been shown to cause strong secretion defects. Here, we show that point mutations introduced at specific locations near the middle or N terminus of the EspP -helix that perturb folding also impair secretion, but to a lesser degree. Surprisingly, we found that even multiple mutations that potentially abolish the stability of several consecutive rungs of the -helix only moderately reduce secretion efficiency. Although these results provide evidence that the free energy derived from passenger domain folding contributes to secretion efficiency, they also suggest that a significant fraction of the energy required for secretion is derived from another source.