Surface display of proteins by gram-negative bacterial autotransporters

Abstract

Expressing proteins of interest as fusions to proteins of the bacterial envelope is a powerful technique with many biotechnological and medical applications. Autotransporters have recently emerged as a good tool for bacterial surface display. These proteins are composed of an N-terminal signal peptide, followed by a passenger domain and a translocator domain that mediates the outer membrane translocation of the passenger. The natural passenger domain of autotransporters can be replaced by heterologous proteins that become displayed at the bacterial surface by the translocator domain. The simplicity and versatility of this system has made it very attractive and it has been used to display functional enzymes, vaccine antigens as well as polypeptides libraries. The recent advances in the study of the translocation mechanism of autotransporters have raised several controversial issues with implications for their use as display systems. These issues include the requirement for the displayed polypeptides to remain in a translocation-competent state in the periplasm, the requirement for specific signal sequences and "autochaperone" domains, and the influence of the genetic background of the expression host strain. It is therefore important to better understand the mechanism of translocation of autotransporters in order to employ them to their full potential. This review will focus on the recent advances in the study of the translocation mechanism of autotransporters and describe practical considerations regarding their use for bacterial surface display.

Figure 1

Organization and biogenesis of autotransporters. A. The typical organization of an autotransporter (AT) comprising an N-terminal signal sequence (SS), a passenger domain and a translocation unit (TU). The passenger domain includes an N-terminal part that bears the activity of the AT and a C-terminal domain, called the autochaperone domain, which is important for efficient translocation across the outer membrane. The TU also has two distinct domains; the N-terminal region is structured as an α-helix, whereas the C-terminal region is structured as a β-barrel. B. The biogenesis of an AT has four main steps: translocation across the inner membrane, periplasmic transport, insertion into and translocation across the outer membrane and, lastly, processing of the passenger domain.

Figure 2

Mechanistic models of outer membrane translocation by autotransporters. A. Hairpin model: In this model the C-terminal part of the passenger domain inserts itself as an unfolded polypeptide in the barrel of the TU, forming a hairpin. The extracellular folding of the autochaperone domain then pulls the remainder of the passenger domain. B. Threading model. This model is similar to the hairpin model but postulates that the N-terminal part of the passenger domain is inserted first, without hairpin. C. Multimeric model. In this model, multiple TUs are assembling in an oligomer forming a big central pore. Folded and unfolded polypeptides could then cross the outer membrane through this pore. D. Omp85 model. In this model, a number of periplasmic and outer membrane proteins organized around Omp85 are involved in the insertion of the TU and also the translocation across the outer membrane of the passenger domain.