A structurally informed autotransporter platform for efficient heterologous protein secretion and display

Abstract

BackgroundThe 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 β-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.ResultsBased on the Hbp crystal structure, five passenger side domains were selected and one by one replaced by ESAT6, whereas a β-helical core structure (β-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 β-stem appeared unstable after translocation, demonstrating the importance of an intact β-stem. By interrupting the cleavage site between passenger and β-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.ConclusionsWe 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.

Decoration of Outer Membrane Vesicles with Multiple Antigens by Using an Autotransporter Approach

“…All plasmids were based on pEH3-HbpD(⌬BamHI) (34), in which the sequence encoding the passenger-␤-domain junction has been altered to prevent autocatalytic intradomain cleavage. For expression of Hbp lacking side domain d1, we made use of a previously described derivative of pEH3-HbpD(⌬BamHI), pHbp(⌬d1) (26), in which the sequence encoding d1 has been substituted for Gly/Ser-encoding linker sequences containing SacI and BamHI restriction sites. HbpD-ESAT6, a chimera in which d1 of Hbp was replaced by the M. tuberculosis antigen ESAT6, was expressed from plasmid pHbpD(⌬d1)-ESAT6 (26).…”

“…For expression of Hbp lacking side domain d1, we made use of a previously described derivative of pEH3-HbpD(⌬BamHI), pHbp(⌬d1) (26), in which the sequence encoding d1 has been substituted for Gly/Ser-encoding linker sequences containing SacI and BamHI restriction sites. HbpD-ESAT6, a chimera in which d1 of Hbp was replaced by the M. tuberculosis antigen ESAT6, was expressed from plasmid pHbpD(⌬d1)-ESAT6 (26). In this plasmid, a SacI/BamHI-flanked E. coli codon-optimized fragment corresponding to M. tuberculosis esxA was inserted into the SacI and BamHI sites of pHbp(⌬d1).…”

“…The mature Hbp passenger folds into an ϳ100-Å ␤-helical stem structure that functions as a stable scaffold for five protruding side domains (d1 to d5) (32). We showed that these side domains are dispensable for translocation and that they can be replaced by heterologous polypeptides (26). In combination with a mutation in the ␤-domain that prevents release of the passenger domain (Fig.…”

“…In combination with a mutation in the ␤-domain that prevents release of the passenger domain (Fig. 1A) (33)(34)(35), optimal exposure of heterologous proteins can be achieved at some distance from the cell surface (26).…”

“…We recently engineered the Escherichia coli autotransporter (AT) hemoglobin protease (Hbp) into a platform for high-density surface display of heterologous proteins in Gram-negative bacteria (26). The AT pathway is one of the mechanisms that have evolved in Gram-negative bacteria to transfer proteins across their complicated cell envelopes, and it is used for secretion of large virulence factors (27).…”