Bacterial display

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Bacterial display (or bacteria display or bacterial surface display) is a protein engineering technique used for in vitro protein evolution. Libraries of polypeptides displayed on the surface of bacteria can be screened using flow cytometry or iterative selection procedures (biopanning). This protein engineering technique allows us to link the function of a protein with the gene that encodes it. Bacterial display can be used to find target proteins with desired properties and can be used to make affinity ligands which are cell-specific. This system can be used in many applications including the creation of novel vaccines, the identification of enzyme substrates and finding the affinity of a ligand for its target protein.

Contents

Bacterial display is often coupled with magnetic-activated cell sorting (MACS) or fluorescence-activated cell sorting (FACS) techniques. Competing methods for protein evolution in vitro are phage display, ribosome display, yeast display, and mRNA display. Bacteriophage display is the most common type of display system used [1] although bacterial display is becoming increasingly popular as technical challenges are overcome. Bacterial display combined with FACS also has the advantage that it is a real-time technique.

History

Cell display systems were first used in 1985, when peptides were genetically fused with proteins displayed on the M13 bacteriophage. Bacteriophage display is a commonly used cell display system, although it carries limitations in the size of proteins that can be displayed. Bacterial display was then introduced in 1986, allowing the surface display of larger proteins. Bacterial display systems were first introduced by Freudl et al. and Charbit et al. in 1986, when they used bacterial surface proteins OmpA and LamB to display peptides. Freudl et al. fused peptides with linkers with the ompA gene, causing the peptides to be expressed in the OmpA proteins. They showed that the proteins were now subject to cleavage by proteinase K. The non-OmpA peptides inserted were therefore a target of proteinase K. Insertion of the foreign peptides did not affect bacterial cell growth. Charbit et al. firstly defined the areas of the LamB protein that were "permissive" for foreign petide insertion (ie that did not lead to a complete loss of functionality of the protein). Then, they explored the versatility of the permissive sites (size limit, nature of the epitope,...) that were all located in surface-exposed loops of the trimeric outer membrane porin, aiming at developing multivalent live bacterial vaccines (12-15). This was the first evidence of using bacterial surface display techniques to express proteins on the surface of cells, without altering the function of the cell. [2]

Principle

Peptides are very useful as therapeutic and diagnostic substances. Their use is getting more popular, and display systems offer a useful way to engineer peptides and optimise their binding capabilities. Cells express surface proteins which can be involved in a whole host of responses including recognition of other cells, interaction with other cells, and cell signalling. Many types of bacteria have cell surface proteins such as the enteropathogenic E. coli intimin protein which is involved in binding to host cells, or the OmpA protein of E. coli cells which is important in keeping the structure of the outer membrane. [3] Many surface proteins are involved in bacterial cell attachment and invasion of the host cell. By using bacterial display, target proteins on the host cell can be identified. These surface proteins need to first be translocated across the bacterial cell membranes from the cytoplasm to the cell surface. Gram-negative bacteria have an additional periplasmic space, which Gram-positive bacteria lack, so they have a harder task of translocating proteins. The display of heterologous proteins on the bacterial cell surface normally requires the fusion of the protein with a surface protein, called a scaffold.

Autotransporter System (Type V Secretion System) T5SS.svg
Autotransporter System (Type V Secretion System)

Scaffold

Scaffolds are used to display the heterologous protein on the bacterial cell surface. There are various scaffolds which have been used such as outer membrane proteins, fimbriae/flagella proteins and CPX (circularly permuted OmpX). [4] The CPX scaffold allows peptide fusion at both termini of the scaffold.

OMPs are common scaffolds for bacterial display. Proteins can also be displayed on the bacterial cell surface through the use of autotransporters. Autotransporters form part of the type V secretion system. They usually have three domains: leader sequence at the N-terminal; central passenger domain; autotransporter domain at the C-terminal. The heterologous protein is inserted at the passenger domain. [5] Another method of heterologous protein fusion is fusion with fimbriae/flagella, which are filamentous protrusions on the cell surface. There are many fimbriae on mainly Gram-negative bacteria, so displaying proteins on fimbriae is advantageous over some other surface proteins which are less numerous. A disadvantage of using fimbriae is that there is a relatively small insert size limit of 10-30 amino acids. [6]

Flow Cytometer Instrument FACS-toestel.JPG
Flow Cytometer Instrument

Once the heterologous protein has been fused with the bacterial cell surface protein, it is exposed to either an enzyme, a cell (expressing a target protein) or an antibody (usually fluorescently tagged), depending on the application of the experiment. The sample is then passed through a beam of light during FACS, in a very narrow stream of fluid so that only one cell can pass at a time, and the fluorescence emitted is detected. Information on the size of the cell can be obtained by the scattering of light and if binding of the heterologous protein with the target protein/cell has occurred, there will be more fluorescence emitted.

Applications

Bacterial surface display can be used for a variety of applications. These include affinity-based screening, antibody epitope mapping, the identification of peptide substrates, the identification of cell-binding peptides and vaccine generation. [7]

Affinity-based Screening

Screening is used to find the apparent affinities of heterologous proteins displayed on the bacterial cell surface for target proteins. This method is usually combined with FACS, and the addition of a non-fluorescent target protein competitor is beneficial to obtaining more accurate binding affinities. Adding a competitor reduces the chance of target proteins rebinding, which would render the binding affinity less accurate.

Cyclic peptide binders Screening

Cyclic peptides can be successfully displayed on bacterial cell surface. [8] By DNA randomization millions of cyclic peptides displayed on cell surface can be screened against a protein target using high-throughput FACS. [9]

Antibody Epitope Mapping

Antibody epitope mapping is used to find the specificity of an antibody. The epitope (antibody binding site of antigens) is expressed on the bacterial cell surface by expressing a region of the gene encoding the antigen. Flow cytometry with fluorescently-labelled antibodies is used to detect the amount of antibody binding to epitope. [10]

Identification of Peptide Substrates

This can be applied to find the best substrates for proteolytic enzymes. The substrate is displayed on the bacterial cell surface between an affinity ligand and the scaffold, and the kinetics of substrate proteolysis is measured using FACS.

Identification of Cell-binding Peptides

Bacterial display can be used to find peptides which bind to specific cells e.g. breast cancer cells or stem cells. Displayed proteins are fluorescently tagged with GFP, so binding interactions between peptides and target cells can be seen by flow cytometry. Control samples are required in order to measure fluorescence levels in the absence of displayed peptides. Samples are also required which don’t contain displayed peptides, but contain mammalian cells and bacterial cells (including the scaffold).

Vaccine Delivery

Vaccine delivery is a very common application of bacterial surface display. There are two types of live bacterial vaccines that can be made:

  1. Normally pathogenic bacteria are weakened so they are no longer pathogenic.
  2. Commensal or food-grade bacteria which are not pathogenic.

Using bacterial surface display of antigens is a valuable alternative to conventional vaccine design for various reasons, one of them being that the proteins expressed on the bacterial cell surface can act favourably as an adjuvant. Conventional vaccines require the addition of adjuvants. Another advantage of generating vaccines using bacterial display systems is that the whole bacterial cell can be incorporated in the live vaccine [11] Unlike bacteriophage display systems which are generally used in vaccine development to find unknown epitopes, bacterial display systems are used to express known epitopes and the cells act as a vaccine delivery system. [12]


Comparison with phage display

Under similar conditions, selection of bacterial-displayed peptides to model protein streptavidin proved worse. [13]

Related Research Articles

<span class="mw-page-title-main">Antigen</span> Molecule triggering an immune response (antibody production) in the host

In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody or T-cell receptor. The presence of antigens in the body may trigger an immune response. The term antigen originally referred to a substance that is an antibody generator. Antigens can be proteins, peptides, polysaccharides, lipids, or nucleic acids.

<span class="mw-page-title-main">Antibody</span> Protein(s) forming a major part of an organisms immune system

An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the pathogen, called an antigen. Each tip of the "Y" of an antibody contains a paratope that is specific for one particular epitope on an antigen, allowing these two structures to bind together with precision. Using this binding mechanism, an antibody can tag a microbe or an infected cell for attack by other parts of the immune system, or can neutralize it directly.

<span class="mw-page-title-main">DNA vaccine</span> Vaccine containing DNA

A DNA vaccine is a type of vaccine that transfects a specific antigen-coding DNA sequence into the cells of an organism as a mechanism to induce an immune response.

An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that binds to the epitope is called a paratope. Although epitopes are usually non-self proteins, sequences derived from the host that can be recognized are also epitopes.

<span class="mw-page-title-main">Phage display</span> Biological technique to evolve proteins using bacteriophages

Phage display is a laboratory technique for the study of protein–protein, protein–peptide, and protein–DNA interactions that uses bacteriophages to connect proteins with the genetic information that encodes them. In this technique, a gene encoding a protein of interest is inserted into a phage coat protein gene, causing the phage to "display" the protein on its outside while containing the gene for the protein on its inside, resulting in a connection between genotype and phenotype. These displaying phages can then be screened against other proteins, peptides or DNA sequences, in order to detect interaction between the displayed protein and those other molecules. In this way, large libraries of proteins can be screened and amplified in a process called in vitro selection, which is analogous to natural selection.

Opsonins are extracellular proteins that, when bound to substances or cells, induce phagocytes to phagocytose the substances or cells with the opsonins bound. Thus, opsonins act as tags to label things in the body that should be phagocytosed by phagocytes. Different types of things ("targets") can be tagged by opsonins for phagocytosis, including: pathogens, cancer cells, aged cells, dead or dying cells, excess synapses, or protein aggregates. Opsonins help clear pathogens, as well as dead, dying and diseased cells.

Virus-like particles (VLPs) are molecules that closely resemble viruses, but are non-infectious because they contain no viral genetic material. They can be naturally occurring or synthesized through the individual expression of viral structural proteins, which can then self assemble into the virus-like structure. Combinations of structural capsid proteins from different viruses can be used to create recombinant VLPs. Both in-vivo assembly and in-vitro assembly have been successfully shown to form virus-like particles. VLPs derived from the Hepatitis B virus (HBV) and composed of the small HBV derived surface antigen (HBsAg) were described in 1968 from patient sera. VLPs have been produced from components of a wide variety of virus families including Parvoviridae, Retroviridae, Flaviviridae, Paramyxoviridae and bacteriophages. VLPs can be produced in multiple cell culture systems including bacteria, mammalian cell lines, insect cell lines, yeast and plant cells.

<span class="mw-page-title-main">Epitope mapping</span> Identifying the binding site of an antibody on its target antigen

In immunology, epitope mapping is the process of experimentally identifying the binding site, or epitope, of an antibody on its target antigen. Identification and characterization of antibody binding sites aid in the discovery and development of new therapeutics, vaccines, and diagnostics. Epitope characterization can also help elucidate the binding mechanism of an antibody and can strengthen intellectual property (patent) protection. Experimental epitope mapping data can be incorporated into robust algorithms to facilitate in silico prediction of B-cell epitopes based on sequence and/or structural data.

<i>Moraxella catarrhalis</i> Species of bacterium

Moraxella catarrhalis is a fastidious, nonmotile, Gram-negative, aerobic, oxidase-positive diplococcus that can cause infections of the respiratory system, middle ear, eye, central nervous system, and joints of humans. It causes the infection of the host cell by sticking to the host cell using trimeric autotransporter adhesins.

Protein tags are peptide sequences genetically grafted onto a recombinant protein. Tags are attached to proteins for various purposes. They can be added to either end of the target protein, so they are either C-terminus or N-terminus specific or are both C-terminus and N-terminus specific. Some tags are also inserted at sites within the protein of interest; they are known as internal tags.

Adhesins are cell-surface components or appendages of bacteria that facilitate adhesion or adherence to other cells or to surfaces, usually in the host they are infecting or living in. Adhesins are a type of virulence factor.

Immunogenicity is the ability of a foreign substance, such as an antigen, to provoke an immune response in the body of a human or other animal. It may be wanted or unwanted:

Antigenic variation or antigenic alteration refers to the mechanism by which an infectious agent such as a protozoan, bacterium or virus alters the proteins or carbohydrates on its surface and thus avoids a host immune response, making it one of the mechanisms of antigenic escape. It is related to phase variation. Antigenic variation not only enables the pathogen to avoid the immune response in its current host, but also allows re-infection of previously infected hosts. Immunity to re-infection is based on recognition of the antigens carried by the pathogen, which are "remembered" by the acquired immune response. If the pathogen's dominant antigen can be altered, the pathogen can then evade the host's acquired immune system. Antigenic variation can occur by altering a variety of surface molecules including proteins and carbohydrates. Antigenic variation can result from gene conversion, site-specific DNA inversions, hypermutation, or recombination of sequence cassettes. The result is that even a clonal population of pathogens expresses a heterogeneous phenotype. Many of the proteins known to show antigenic or phase variation are related to virulence.

<span class="mw-page-title-main">Maltose-binding protein</span>

Maltose-binding protein (MBP) is a part of the maltose/maltodextrin system of Escherichia coli, which is responsible for the uptake and efficient catabolism of maltodextrins. It is a complex regulatory and transport system involving many proteins and protein complexes. MBP has an approximate molecular mass of 42.5 kilodaltons.

<span class="mw-page-title-main">Immunolabeling</span> Procedure for detection and localization of an antigen

Immunolabeling is a biochemical process that enables the detection and localization of an antigen to a particular site within a cell, tissue, or organ. Antigens are organic molecules, usually proteins, capable of binding to an antibody. These antigens can be visualized using a combination of antigen-specific antibody as well as a means of detection, called a tag, that is covalently linked to the antibody. If the immunolabeling process is meant to reveal information about a cell or its substructures, the process is called immunocytochemistry. Immunolabeling of larger structures is called immunohistochemistry.

<span class="mw-page-title-main">Reverse vaccinology</span> Vaccine development via genomics to find antigens

Reverse vaccinology is an improvement of vaccinology that employs bioinformatics and reverse pharmacology practices, pioneered by Rino Rappuoli and first used against Serogroup B meningococcus. Since then, it has been used on several other bacterial vaccines.

2F5 is a broadly neutralizing human monoclonal antibody (mAb) that has been shown to bind to and neutralize HIV-1 in vitro, making it a potential candidate for use in vaccine synthesis. 2F5 recognizes an epitope in the membrane-proximal external region (MPER) of HIV-1 gp41. 2F5 then binds to this epitope and its constant region interacts with the viral lipid membrane, which neutralizes the virus.

A neutralizing antibody (NAb) is an antibody that defends a cell from a pathogen or infectious particle by neutralizing any effect it has biologically. Neutralization renders the particle no longer infectious or pathogenic. Neutralizing antibodies are part of the humoral response of the adaptive immune system against viruses, intracellular bacteria and microbial toxin. By binding specifically to surface structures (antigen) on an infectious particle, neutralizing antibodies prevent the particle from interacting with its host cells it might infect and destroy.

<span class="mw-page-title-main">Affimer</span> Type of protein

Affimer molecules are small proteins that bind to target proteins with affinity in the nanomolar range. These engineered non-antibody binding proteins are designed to mimic the molecular recognition characteristics of monoclonal antibodies in different applications. These affinity reagents have been optimized to increase their stability, make them tolerant to a range of temperatures and pH, reduce their size, and to increase their expression in E.coli and mammalian cells.

Although cell wall carbohydrates are ideal immunotherapeutic targets due to their abundance in bacteria and high level of conservation, their poor immunogenicity compared with protein targets complicates their use for the development of protective antibodies. A lysibody is a chimeric antibody in which the Fab region is the binding domain from a bacteriophage lysin, or the binding domain from an autolysin or bacteriocin, all of which bind to bacterial cell wall carbohydrate epitopes. This is linked to the Fc of Immunoglobulin G (IgG). The chimera forms a stable homodimer held together by hinge-region disulfide bonds. Thus, lysibodies are homodimeric hybrid immunoglobulin G molecules that can bind with high affinity and specificity to a carbohydrate substrate in the bacterial cell wall peptidoglycan. Lysibodies behave like authentic IgG by binding at high affinity to their bacterial wall receptor, fix complement and therefore promote phagocytosis by macrophages and neutrophils, protecting mice from infection in model systems. Since cell wall hydrolases, autolysins and bacteriocins are ubiquitous in nature, production of lysibodies specific for difficult to treat pathogenic bacteria is possible.

References

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  2. ↑ Freudl R, MacIntyre S, Degen M, Henning U (1986). "Cell Surface Exposure of the Outer Membrane Protein OmpA of Escherichia coli K-12". J Mol Biol . 188 (3): 491–4. doi:10.1016/0022-2836(86)90171-3. PMID   3525847.
  3. ↑ Wang Y (2002). "The Function of OmpA in Escherichia coli". Biochem Biophys Res Commun . 292 (2): 396–401. doi:10.1006/bbrc.2002.6657. PMID   11906175.
  4. ↑ Getz JA, Schoep TD, Daugherty PS (2012). "Peptide Discovery Using Bacterial Display and Flow Cytometry". Protein Engineering for Therapeutics, Part B. Methods Enzymol . Methods in Enzymology. Vol. 503. pp. 75–97. doi:10.1016/B978-0-12-396962-0.00004-5. ISBN   9780123969620. PMID   22230566.
  5. ↑ Wernerus H, Stahl S (2004). "Biotechnological Applications for Surface-engineered Bacteria". Biotechnol Appl Biochem . 40 (Pt 3): 209–28. doi:10.1042/BA20040014. PMID   15035661. S2CID   9395029.
  6. ↑ Klemm P, Schembri MA (2000). "Bacterial Adhesins:Function and Structure". Int J Med Microbiol . 290 (1): 27–35. doi:10.1016/S1438-4221(00)80102-2. PMID   11043979.
  7. ↑ Daugherty PS (2007). "Protein Engineering with Bacterial Display". Curr Opin Struct Biol . 17 (4): 474–80. doi:10.1016/j.sbi.2007.07.004. PMID   17728126.
  8. ↑ Palei, Shubhendu; Becher, Kira S.; Nienberg, Christian; Jose, Joachim; Mootz, Henning D. (2019-01-02). "Bacterial Cell‐Surface Display of Semisynthetic Cyclic Peptides". ChemBioChem. 20 (1): 72–77. doi:10.1002/cbic.201800552. ISSN   1439-4227. PMID   30216604. S2CID   52277462.
  9. ↑ Palei, Shubhendu; Jose, Joachim; Mootz, Henning D. (2022), Coppock, Matthew B.; Winton, Alexander J. (eds.), "Preparation of Bacterial Cell-Surface Displayed Semisynthetic", Peptide Macrocycles, New York, NY: Springer US, vol. 2371, pp. 193–213, doi:10.1007/978-1-0716-1689-5_11, ISBN   978-1-0716-1688-8, PMID   34596850, S2CID   238237296 , retrieved 2022-05-12
  10. ↑ Rockberg J, Lofblom J, Hjelm B, Uhlen M, Stahl S (2008). "Epitope Mapping of Antibodies Using Bacterial Surface Display". Nature Methods . 5 (12): 1039–45. doi:10.1038/nmeth.1272. PMID   19029907. S2CID   12078882.
  11. ↑ Westerlund-Wikstrom B (2000). "Peptide Display on Bacterial Flagella: Principles and Applications". Int J Med Microbiol . 290 (3): 223–30. doi:10.1016/S1438-4221(00)80119-8. PMID   10959724.
  12. ↑ Benhar I (2001). "Biotechnological Applications of Phage and Cell Display". Biotechnology Advances . 19 (1): 1–33. doi:10.1016/S0734-9750(00)00054-9. PMID   14538090.
  13. ↑ Lunder, et al. (2005). "Comparison of Bacterial and Phage Display Peptide Libraries in Search of Target-Binding Motif". Applied Biochemistry and Biotechnology. 127 (2): 125–131. doi:10.1385/ABAB:127:2:125. PMID   16258189. S2CID   45243314.

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