Surface display of Salmonella epitopes in Escherichia coli and Staphylococcus carnosus

Abstract

BackgroundSalmonella enterica serotype Enteritidis (SE) is considered to be one of the most potent pathogenic Salmonella serotypes causing food-borne disease in humans. Since a live bacterial vaccine based on surface display of antigens has many advantages over traditional vaccines, we have studied the surface display of the SE antigenic proteins, H:gm and SefA in Escherichia coli by the β-autotransporter system, AIDA. This procedure was compared to protein translocation in Staphylococcus carnosus, using a staphylococci hybrid vector earlier developed for surface display of other vaccine epitopes.ResultsBoth SefA and H:gm were translocated to the outer membrane in Escherichia coli. SefA was expressed to full length but H:gm was shorter than expected, probably due to a proteolytic cleavage of the N-terminal during passage either through the periplasm or over the membrane. FACS analysis confirmed that SefA was facing the extracellular environment, but this could not be conclusively established for H:gm since the N-terminal detection tag (His6) was cleaved off. Polyclonal salmonella antibodies confirmed the sustained antibody-antigen binding towards both proteins. The surface expression data from Staphylococcus carnosus suggested that the H:gm and SefA proteins were transported to the cell wall since the detection marker was displayed by FACS analysis.ConclusionApart from the accumulated knowledge and the existence of a wealth of equipment and techniques, the results indicate the selection of E. coli for further studies for surface expression of salmonella antigens. Surface expression of the full length protein facing the cell environment was positively proven by standard analysis, and the FACS signal comparison to expression in Staphylococcus carnosus shows that the distribution of the surface protein on each cell was comparatively very narrow in E. coli, the E. coli outer membrane molecules can serve as an adjuvant for the surface antigenic proteins and multimeric forms of the SefA protein were detected which would probably be positive for the realisation of a strong antigenic property. The detection of specific and similar proteolytic cleavage patterns for both the proteins provides a further starting point for the investigation and development of the Escherichia coli AIDA autotransporter efficiency.

Surface Expression of ω-Transaminase in Escherichia coli

“…Therefore, proteolysis occurs either during transit through the periplasm, during surface translocation, or at the cell surface. We previously expressed the synthetic protein Z derived from Staphylococcus aureus protein A (21) and the Salmonella proteins SefA and H:gm (13,21) and observed similar proteolysis patterns. Together with the results in the present study, we observe that larger recombinant passenger proteins appear more susceptible to proteolysis.…”

“…The host organism for surface expression was an OmpT-negative strain derived from E. coli K-12 0:17 (20) (0:17⌬OmpT), which has previously been found suitable for recombinant surface expression using the AIDA autotransporter (13,18,21). E. coli strain BL21(DE3) was used for production of purified transaminase using the T7-based promoter of pET28aϩ.…”

“…The present understanding of the mechanism has been extensively reviewed elsewhere (8,9). The adhesin involved in diffuse adherence (AIDA-I) of enteropathogenic Escherichia coli (10) is one of the most frequently used autotransporters for recombinant surface expression, and its use has previously been reported for display of enzymes (11), enzyme inhibitors (12), and vaccine epitopes (13).…”

Single-cell Characterization of Autotransporter-mediated Escherichia coli Surface Display of Disulfide Bond-containing Proteins

“…The utility of surface display of functional peptides and proteins for biotechnological applications has been demonstrated in several different contexts including: wholecell biocatalysis based on esterases (1), bioremediation using recombinant display of organophosphorous hydrolases and metallothioneins (2,3), glucose-responsive biosensors (4), and protein engineering of displayed libraries (5,6). In the context of surface display of antigenic recombinant peptides/proteins for delivery of live vaccines, besides the obvious safety advantage of using surface display on nonpathogenic bacteria, there are several other benefits such as the cost and ease of manufacturing, the ability of bacterial components such as lipopolysaccharide (LPS) to function as adjuvants to stimulate the immune system via Toll-like receptors leading to sustained immunity (7), and the ability of the mammalian innate immune system to recognize prokaryotic mRNA (present only in live cells) leading to protective immunity (8).…”

“…Similarly, biotechnological applications that involve displaying libraries of protein molecules require knowledge of the number of functional recombinant protein molecules displayed on every single cell. Although flow cytometry has been previously applied to study of ATs, these have been predominantly restricted to quantifying epitope tags that indicate surface protein display and not functional protein display (7,19,30).…”