Autotransporter-based cell surface display in Gram-negative bacteria

Abstract

Cell surface display of proteins can be used for several biotechnological applications such as the screening of protein libraries, whole cell biocatalysis and live vaccine development. Amongst all secretion systems and surface appendages of Gram-negative bacteria, the autotransporter secretion pathway holds great potential for surface display because of its modular structure and apparent simplicity. Autotransporters are polypeptides made up of an N-terminal signal peptide, a secreted or surface-displayed passenger domain and a membrane-anchored C-terminal translocation unit. Genetic replacement of the passenger domain allows for the surface display of heterologous passengers. An autotransporter-based surface expression module essentially consists of an application-dependent promoter system, a signal peptide, a passenger domain of interest and the autotransporter translocation unit. The passenger domain needs to be compatible with surface translocation although till now no general rules have been determined to test this compatibility. The autotransporter technology for surface display of heterologous passenger domains is critically discussed for various applications.

Genome-Wide Analyses Reveal Genes Subject to Positive Selection in Pasteurella multocida

“…Autotransporter proteins are typically VFs in P. multocida (Nicolay et al, 2015). The gene icsA (OG1421) encoding an autotransporter related to the Type V secretion system, was undergoing significantly positive selection (FDR = 0.002) (Table S7).…”

“…It was worth noting that intragenic homologous recombination also occurred on icsA (2,556 nt), with one breakpoint at position 331 (Table S6). The outer membrane protein IcsA of P. multocida harbors a β-barrel domain (PF03797) at the C-terminal followed by an α-helical linker essential for outer membrane translocation of the passenger domain pertactin (PF03212) with characteristic β-helix (Nicolay et al, 2015). Structural mapping of seven positively selected sites on IcsA showed that the majority of sites (i.e., residues 159, 220, 315, 481, and 690) were located at the loops connecting the β-strands (Figure 6).…”

“…Protein secondary structure is color-coded based on the predicted PFAM domains and signal peptide cleavage site as well as modular organization previously described by Nicolay et al (2015): an N-terminal signal peptide in red, a secreted mature passenger domain in green, and a C-terminal translocator domain in cyan with an upstream α-helical linker in purple. Orange spheres stand for amino acid residues that are subject to strong positive selection (posterior probability > 95%).…”

Escherichia coli surface display for the selection of nanobodies

“…The limitations of the above systems led us to explore alternative E. coli display systems for the selection of Nbs (and perhaps other Ab fragments). An important clue came from the study of a large family of proteins secreted by Gram‐negative bacteria called autotransporters (ATs; Henderson et al ., ; Nicolay et al ., ). Classical ATs contain within their sequence a C‐terminal β‐barrel, comprising of 12 antiparallel β‐strands and an internal α‐helix (Oomen et al ., ; van den Berg, ; Gawarzewski et al ., ), which acts as a ‘helper’ domain assisting the translocation of the N‐terminal portion of the polypeptide across the OM.…”

Autotransporter Adhesins in Escherichia coli Pathogenesis

“…This research has led to the use of AT proteins as vaccine candidates, whereby pertactin from Bordetella pertussis or NadA from Neisseria meningitidis are constituents of human vaccines . Furthermore, the seemingly simple AT secretion system has also attracted attention for biotechnological applications as a carrier of heterologous proteins to the bacterial cell surface . This review describes the AIDA‐I type ATs in E. coli strains, which are representative of the great diversity of AT adhesins in Gram‐negative bacteria.…”