Gram negative promiscuous lipoproteins keep surface topology when transplanted from one species to another and can deliver foreign polypeptides to the bacterial surface

ABSTRACT: In Gram negative bacteria outer membrane-associated lipoproteins caneither face the periplasm orprotrude out of the bacterial surface. The mechanisms involved in lipoprotein transport through the outer membrane are not fully elucidated. A group of lipoproteins reach the surface by using species-specific transport machineries. By contrast, a still poorly characterized group, we referred to as “promiscuous lipoproteins”, appears to cross the outer membrane using transport systems conserved among diderms. To provide further evidence of the existence of promiscuous lipoproteins,we tested the expression and compartmentalization in E. coli of three surface-exposed lipoproteins, twofrom Neisseria meningitidis (Nm-fHbp and NHBA) andone fromAggregatibacteractinomycetemcomitans (Aa-fHbp). We found that the three lipoproteins werelipidated and compartmentalized in E. coliouter membrane and in Outer Membrane Vesicles (OMVs). Furthermore, FACS analysis,proteolytic surface shaving, and confocal microscopy revealed that the three proteins were also exposed on the external side 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 external side of the outer membrane. Heterologous polypeptides fused to the C termini of the proteins are efficiently transported to the E. coli surface and efficiently compartmentalized in OMVs. These data indicate that promiscuous lipoproteins travel from the cytoplasm to the cell surface using conserved mechanisms and when transplanted from one species to another they maintain their natural topology without the need of ancillary transport machineries. Furthermore such proteins can be exploited in biotechnological applications requiring the decoration of Gram negative bacterial surface with foreign polypeptides.

INSTRUMENT(S): ultraflex

ORGANISM(S): Escherichia Coli

TISSUE(S): Cell Suspension Culture

SUBMITTER: Luca Bini  

LAB HEAD: Luca Bini

PROVIDER: PXD005732 | Pride | 2017-05-11

REPOSITORIES: Pride