Some Gram-negative Lipoproteins Keep Their Surface Topology When Transplanted from One Species to Another and Deliver Foreign Polypeptides to the Bacterial Surface

Abstract

In Gram-negative bacteria, outer membrane-associated lipoproteins can either face the periplasm or protrude out of the bacterial surface. The mechanisms involved in lipoprotein transport through the outer membrane are not fully elucidated. Some lipoproteins reach the surface by using species-specific transport machinery. By contrast, a still poorly characterized group of lipoproteins appears to always cross the outer membrane, even when transplanted from one organism to another. To investigate such lipoproteins, we tested the expression and compartmentalization in E. coli of three surface-exposed lipoproteins, two from Neisseria meningitidis (Nm-fHbp and NHBA) and one from Aggregatibacter actinomycetemcomitans (Aa-fHbp). We found that all three lipoproteins were lipidated and compartmentalized in the E. coli outer membrane and in outer membrane vesicles. Furthermore, fluorescent antibody cell sorting analysis, proteolytic surface shaving, and confocal microscopy revealed that all three proteins were also exposed on the surface of the outer membrane. Removal or substitution of the first four amino acids following the lipidated cysteine residue and extensive deletions of the C-terminal regions in Nm-fHbp did not prevent the protein from reaching the surface of the outer membrane. Heterologous polypeptides, fused to the C termini of Nm-fHbp and NHBA, were efficiently transported to the E. coli cell surface and compartmentalized in outer membrane vesicles, demonstrating that these lipoproteins can be exploited in biotechnological applications requiring Gram-negative bacterial surface display of foreign polypeptides.

Synergistic Protective Activity of Tumor-Specific Epitopes Engineered in Bacterial Outer Membrane Vesicles

“…We first tested whether OMVs decorated with the Nm-fHbp-vIII fusion protein carrying three copies of EGFRvIIIpep at its C-terminus could induce anti-EGFRvIIIpep antibodies and whether such anti-EGFRvIIIpep immune response could protect mice from B16F10EGFRvIII challenge. The expression of EGFRvIIIpep in the OMVs from E. coli BL21 ΔompA strain has been recently described ( 20 ). Briefly, a synthetic DNA encoding three copies of EGFRvIIIpep was fused to the 3′ end of the Neisseria meningitidis fHbp gene, thus generating a chimera (Nm-fHbp-vIII) constituted of the full length fHbp protein and the EGFRvIII tri-peptide attached to its C-terminus (Figure 1 A).…”

“…The construction of pET21-Nm-fHbp and pET-Nm-fHbp-vIII plasmids expressing the Neisseria meningitidis fHbp and fHbp fused to three repeated copies of EGFRvIII peptide, respectively, was previously described ( 20 ). pET-Nm-fHbp-M30vIII plasmid carries the N. meningitidis fHbp gene fused to a synthetic DNA fragment encoding three copies of B16-M30 peptide and three copies of EGFRvIII peptide, each copy intercalated by a Glycine–Serine (GS) spacer (Figure 4 A).…”

“…Recently, we showed that different bacterial antigens could be delivered to the lumen of E. coli vesicles by fusing their coding sequences to a leader peptide for secretion ( 19 ). Moreover, we showed that heterologous lipoproteins could be incorporated into the OMV membrane and that such proteins could serve as chaperones to transport heterologous polypeptides to the OMV surface ( 20 ). Third, OMVs can be rapidly and easily purified from bacterial culture supernatant.…”

Decorating the Surface of Escherichia Coli with Bacterial Lipoproteins: A Comparative Analysis of Different Display Systems

“…However, cells from the same culture frequently showed two different populations in which a speci c gene is either surface-exposed or not. This behaviour may be due to the occurrence of bistability [43] [44]. As possible consequence of this, the percentage of expressing cells showed a signi cant variation between cultures.…”

“…This behaviour may be due to the occurrence of bistability, in which cells with the same genotype separate into two populations, either expressing or not a speci c gene. One explanation could be related to expression levels signi cantly higher than that characteristic of physiological conditions leading to an overloading of the folding machinery and the tra cking systems, thus preventing the correct exposure of the protein on the surface [41][42][43] [44]. An intriguing aspect that is worth noting is that the percentage of viable and not aggregated bacterial cells accounts for up to 93% of the population (Additional le 4: Figure S1, panel A),…”