Summary auto-generated
Fimbriae are hair-like surface structures on bacteria that function as adhesins and can be engineered to display heterologous peptides. This review describes how fimbriae serve as platforms for surface display of foreign antigens, with type 1 fimbriae—found on E. coli and other Enterobacteriaceae—representing the best-characterized system. The major structural protein (FimA) and tip protein (FimH) are the primary targets for inserting foreign sequences. Type 1 fimbriae can display diverse epitopes from pathogenic viruses and other organisms, with high valency (hundreds of thousands of copies per cell), strong immunogenicity, and ease of purification making them attractive vaccine candidates. Beyond vaccines, fimbriae have been adapted to create random peptide libraries for identifying sequences with specific binding properties, such as to heavy metals. The architecture of fimbriae resembles filamentous phage displays, with the minor component FimH accommodating larger inserts than the major protein FimA. Multiple fimbrial types—including P, K88, CS31A, and type 4 fimbriae—have been successfully used for epitope display across different bacterial species and applications.
Key findings
- Type 1 fimbriae can be engineered to display foreign peptide epitopes in either the major protein (FimA) at high valency or in the adhesin protein (FimH) with preserved binding function
- Fimbriae-based display systems have been used to create successful recombinant vaccines against viral and bacterial pathogens, including proof-of-concept demonstrations in animal models
- Random peptide libraries inserted into FimH have been screened to identify sequences with specific binding properties to metals and metal oxides using bacterial surface display
- Multiple fimbrial types from different bacteria can accommodate heterologous inserts, though constraints exist regarding insert size, hydrophobicity, and cysteine content
- Fimbriae offer advantages over other bacterial display systems due to extremely high copy number (up to 500,000 copies per cell), strong immunogenicity, and ease of large-scale purification
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Abstract
The double membrane system of Gram-negative bacteria is an efficient barrier instrumental in maintaining a steady environment in the cytoplasm. Only a few low-molecular-mass substances are able to cross this barrier unassisted from the cell exterior. On the other hand, the Gram-negative cell envelope also constitutes a formidable obstacle for proteins destined for the cell exterior. Gram-negative bacteria have developed various systems for protein export to the cell exterior; four major systems can be distinguished (Lory, 1998 ). By these systems a wide range of proteins can be routed to the surface, and many of these remain physically attached to and displayed on the cell surface. Such proteins are key players in a number of natural processes, such as adhesion, colonization of target surfaces, biofilm formation, motility, signal transduction, enzymic degradations, etc. However, many proteins of interest in medicine and biotechnology are not surface-displayed and/or not of bacterial origin. An attractive way to obtain surface display of such proteins or sectors thereof is to graft them into permissible positions on a naturally occurring bacterial surface protein and express the chimeric protein on the cell surface. Surface display of heterologous proteins on bacteria has resulted in a number of applications, such as recombinant vaccines, reagents for diagnostics, whole-cell biocatalysts and bioadsorbants, and systems for scanning peptide libraries (Georgiou et al., 1997 ).
Fimbriae are adhesive bacterial organelles which enable bacteria to target and to colonize specific host tissues (for reviews, see Klemm, 1994 ). They are long, thread-like surface structures, found in up to about 500 copies per cell. A large diversity of fimbriae, mostly of Gram-negative origin, are known. In Gram-negative bacteria, fimbriae are in most cases assembled via the chaperone/usher pathway. The initial translocation of organelle components across the cytoplasmic membrane is dependant on the normal type II export system. However, further export from the periplasm to the cell exterior is mediated by a specific two-component system consisting of a periplasmic chaperone and an usher, an outer-membrane-located pore, which serves as assembly platform (Hultgren et al., 1996 ; Klemm & Schembri, 2000 ). A highly choreographed series of specific molecular interactions ultimately leads to the formation of the fimbrial organelle, a polymeric structure in which hundreds of subunits are held together by non-covalent subunitsubunit interactions. Specific motifs present on the structural proteins are involved in interactions with the transport machinery and subunitsubunit interactions; these are obviously non-permissible regions for heterologous insertion.
This review primarily deals with display systems based on type 1 fimbriae. These can be considered as paradigms for fimbrial display, firstly because type 1 fimbriae are the best structurally characterized fimbrial system and are very representative of this type of organelle; secondly, because most display studies have been carried out with this system. Type 1 fimbriae are found on the majority of Enterobacteriaceae including Escherichia coli. A single type 1 fimbria is a thin, 7 nm wide and approximately 1 µm long surface polymer. The bulk of the organelle is made up of about 1000 subunits of the major building element, the FimA protein, stacked in a helical cylinder (Brinton, 1965 ). Additionally, small amounts of minor components are present as integral, primarily tip-located constituents (Krogfelt & Klemm, 1988 ; Jones et al., 1995 ). The minor components, viz. FimF, FimG and FimH, are involved in initiation of organelle synthesis and consequently in length regulation (Klemm & Christiansen, 1987 ; Russell & Orndorff, 1992 ). The FimH protein has been shown to be the actual receptor-binding molecule which recognizes D-mannose-containing structures (Krogfelt et al., 1990 ). The FimF and FimG components seem to be required for integration of the FimH adhesin into the fimbriae. The specific export system consists of the FimC chaperone and the FimD usher proteins (Klemm, 1992 ; Klemm & Christiansen, 1990 ).
The biogenesis of type 1 fimbriae has been shown to be quite adaptable with regard to incorporation of homologous structural components and their assembly into fimbriae. Thus, components from F1C fimbriae exhibiting as little as 34% identity were readily incorporated into type 1 fimbriae, resulting in hybrid organelles (Klemm et al., 1994 ). This and other structurefunction information suggested that type 1 fimbriae could be used as carriers of heterologous sequences. Two structural components of type 1 fimbriae have been used to display foreign peptides, viz. FimA and FimH. In both cases a strategy employing in-frame fusion of the heterologous sequences into permissible sites has been used. If insert positions are chosen which do not interfere with the bioassembly of the organelles normal fimbriae will result (Fig. 1).