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Looks can be deceiving: recent insights into the mechanism of protein secretion by the autotransporter pathway.

ABSTRACT: Autotransporters are a large superfamily of cell surface proteins produced by Gram-negative bacteria that consist of an N-terminal extracellular domain ('passenger domain') and a C-terminal ?-barrel domain that resides in the outer membrane (OM). Although it was originally proposed that the passenger domain is translocated across the OM through a channel formed exclusively by the covalently linked ?-barrel domain, this idea has been strongly challenged by a variety of observations. Recent experimental results have suggested a new model in which both the translocation of the passenger domain and the membrane integration of the ?-barrel domain are facilitated by the Bam complex, a highly conserved heteroligomer that plays a general role in OM protein assembly. Other factors, including periplasmic chaperones and inner membrane proteins, have also recently been implicated in the biogenesis of at least some members of the autotransporter superfamily. New results have raised intriguing questions about the energetics of the secretion reaction and the relationship between the assembly of autotransporters and the assembly of other classes of OM proteins. Concomitantly, new mechanistic and structural insights have expanded the utility of the autotransporter pathway for the surface display of heterologous peptides and proteins of interest.

SUBMITTER: Bernstein HD

PROVIDER: S-EPMC6376861 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Looks can be deceiving: recent insights into the mechanism of protein secretion by the autotransporter pathway.

Bernstein Harris D HD

Molecular microbiology 20150515 2

Autotransporters are a large superfamily of cell surface proteins produced by Gram-negative bacteria that consist of an N-terminal extracellular domain ('passenger domain') and a C-terminal β-barrel domain that resides in the outer membrane (OM). Although it was originally proposed that the passenger domain is translocated across the OM through a channel formed exclusively by the covalently linked β-barrel domain, this idea has been strongly challenged by a variety of observations. Recent experi ...[more]

PMID: 25881492

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Project description:BACKGROUND: The self-sufficient autotransporter (AT) pathway, ubiquitous in Gram-negative bacteria, combines a relatively simple protein secretion mechanism with a high transport capacity. ATs consist of a secreted passenger domain and a ?-domain that facilitates transfer of the passenger across the cell-envelope. They have a great potential for the extracellular expression of recombinant proteins but their exploitation has suffered from the limited structural knowledge of carrier ATs. Capitalizing on its crystal structure, we have engineered the Escherichia coli AT Hemoglobin protease (Hbp) into a platform for the secretion and surface display of heterologous proteins, using the Mycobacterium tuberculosis vaccine target ESAT6 as a model protein. RESULTS: Based on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a ?-helical core structure (?-stem) was left intact. The resulting Hbp-ESAT6 chimeras were efficiently and stably secreted into the culture medium of E. coli. On the other hand, Hbp-ESAT6 fusions containing a truncated ?-stem appeared unstable after translocation, demonstrating the importance of an intact ?-stem. By interrupting the cleavage site between passenger and ?-domain, Hbp-ESAT6 display variants were constructed that remain cell associated and facilitate efficient surface exposure of ESAT6 as judged by proteinase K accessibility and whole cell immuno-EM analysis. Upon replacement of the passenger side domain of an alternative AT, EspC, ESAT6 was also efficiently secreted, showing the approach is more generally applicable to ATs. Furthermore, Hbp-ESAT6 was efficiently displayed in an attenuated Salmonella typhimurium strain upon chromosomal integration of a single encoding gene copy, demonstrating the potential of the Hbp platform for live vaccine development. CONCLUSIONS: We developed the first structurally informed AT platform for efficient secretion and surface display of heterologous proteins. The platform has potential with regard to the development of recombinant live vaccines and may be useful for other biotechnological applications that require high-level secretion or display of recombinant proteins by bacteria.

Dataset Information

Looks can be deceiving: recent insights into the mechanism of protein secretion by the autotransporter pathway.

Publications

Looks can be deceiving: recent insights into the mechanism of protein secretion by the autotransporter pathway.

Similar Datasets

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