Characterization of adhesive epitopes with the OmpS display system

Abstract

OmpS is an outer membrane protein of Vibrio cholerae where it forms trimeric pores that function in the uptake of maltose and maltodextrins. Based on sequence similarity to LamB proteins, a model of OmpS folding in the outer membrane has been constructed. According to this model, OmpS contains 18 transmembrane b-strands and nine surface-accessible loops. Adhesive epitopes can, when inserted into surface-accessible loop 4 (L4) and expressed in Escherichia coli, retain their functional characteristics. We inserted three d-repeats from the Staphylococcus aureus fibronectin-binding protein FnBPA into L4 of OmpS and showed that E. coli cells expressing these hybrids bind fibronectin. DNA fragments covering the N-terminal half of the globoside-binding P-fimbrial adhesin class II PapG of E. coli were cloned into the same surface accessible loop (L4) of OmpS. Fragments of papG encoding 53 or 186 amino acids from the N-terminal end of class II PapG adhesin were found to confer bacterial adhesiveness to globoside. Removal of 23 amino acids from the N-terminus of PapG did not affect receptor binding, but removal of 31 amino acids abolished it. The newly developed night sky image technique was also used to demonstrate the binding properties of membrane vesicles carrying the hybrid proteins. We raised antibodies against the purified hybrid protein containing 53 amino acids from PapG. This antiserum recognized the P-fimbriae on E. coli cells. These data provide evidence that the N-terminal first 53 amino acids of class II PapG contain the receptor-binding domain.

Novel Bacterial Membrane Surface Display System Using Cell Wall-Less L-Forms of Proteus mirabilis and Escherichia coli

“…3). In combination with solubilization experiments, these results gave proof that the fusion proteins were embedded in the CM and did not form considerable amounts of aggregates as described for other surface display systems (22).…”

“…In gram-positive bacteria, surface anchors have been derived from lipoproteins, cell wall proteins, or S-layer proteins (24,32,33). However, depending on the displayed protein and the desired application, each system has its own advantages and disadvantages, such as the size limitation of the displayed protein, mislocalization or formation of inclusion bodies, association with lipopolysaccharides (LPS), or destabilization of the outer membrane (10,17,22). There is still a need for further developments to increase the repertoire of applications of surface display systems (21).…”

Cell‐Surface display of heterologous proteins: From high‐throughput screening to environmental applications

“…Methods for the display of recombinant proteins on the surface of Gram-negative bacteria have been the subject of several recent reviews (Earhart, 2000;Georgiou et al, 1997;Lang et al, 2000;Westerlund-Wikstrom, 2000). Therefore, we will present only the salient features of different strategies for protein display, emphasizing features relevant to cell surface engineering.…”

“…For this reason it has been necessary to exploit native, integral outer membrane proteins as vehicles for display purposes. The utility of most major E. coli outer membrane proteins has been evaluated (for recent examples, see Camaj et al, 2001;Chang et al, 1999;Etz et al, 2001;Lang, 2000;Lang et al, 2000;Xu and Lee, 1999). These include LamB, OmpA, OmpC, OmpS, FhuA, the lipoprotein TraT and others for cell surface engineering.…”

Identification of Vibrio harveyi proteins involved in the specific immune response of Senegalese sole ( Solea senegalensis , Kaup)

“…In particular, it is indispensable for the diffusion of large solutes such as maltooligosaccharides. The immunologic role is unknown in V. harveyi but it is well-known in other Vibrio species such as Vibrio cholerae (protein OmpS) [13]. In addition, the potentiality of this protein as vaccine antigen in V. harveyi can be borne out if we consider that protective immune response against Aeromonas hydrophila and Edwardsiella tarda has been reported after immunization with purified recombinant A. hydrophila maltoporine [14].…”