Bacteriophage Capsid Modification by Genetic and Chemical Methods

Abstract

Bacteriophages are viruses whose ubiquity in nature and remarkable specificity to their host bacteria enable an impressive and growing field of tunable biotechnologies in agriculture and public health. Bacteriophage capsids, which house and protect their nucleic acids, have been modified with a range of functionalities (e.g. fluorophores, nanoparticles, antigens, drugs) to suit their final application. Functional groups naturally present on bacteriophage capsids can be used for electrostatic adsorption or bioconjugation but their impermanence and poor specificity can lead to inconsistencies in coverage and function. To overcome these limitations, researchers have explored both genetic and chemical modifications to enable strong, specific bonds between phage capsids and their target conjugates. Genetic modification methods involve introducing genes for alternative amino acids, peptides, or protein sequences into either the bacteriophage genomes or capsid genes on host plasmids to facilitate recombinant phage generation. Chemical modification methods rely on reacting functional groups present on the capsid with activated conjugates under the appropriate solution pH and salt conditions. This review surveys the current state-of-the-art in both genetic and chemical bacteriophage capsid modification methodologies, identifies major strengths and weaknesses of methods, and discusses areas of research needed to propel bacteriophage technology in development of biosensors, vaccines, therapeutics, and nanocarriers.

1. INTRODUCTION

Bacteriophages (phages) have played an instrumental role in biotechnology since their discovery in the late 1890’s, including being utilized for antibacterial therapy, vaccines, and gene delivery vehicles.^1,2 Phages are viruses that infect bacteria within a typically narrow host range, and are non-infectious towards non-bacterial cells. Following recognition of their host and injection of their genetic material, phages take over the host bacterium’s cellular machinery to propagate tens to thousands of more phages, lysing the host cell to release newly synthesized phage progeny into the environment. Due to this remarkable orders-of-magnitude replication cycle, phages are highly abundant (in fact, are reported to be the most abundant organism on Earth)³ and have adapted to survive in a variety of environments across the globe including marine waters,^4–6 human bodies,^7–9 and diverse soils.^10–12 Phages can be propagated with standard lab glassware and equipment or in bioreactors for large-scale production.^13–15

Wild type phages are employed as therapeutics, bioreceptors, and biocontrol agents. Several recent cases have demonstrated that patients infected with multi-drug resistant bacteria including Staphylococcus aureus,^16–18 Acinetobacter baumannii,^19–21 and Pseudomonas aeruginosa^22–24 have successfully recovered after phage administration. Commercial phage preparations can be applied to food products and contact surfaces to help control contamination by foodborne pathogens.^25–27 Biosensors relying on phages as bacteria biorecognition elements have allowed for the rapid detection of pathogens such as Salmonella Typhimurium and Staphylococcus aureus.^28,29

Although phages offer several benefits, biological limitations of phage structural features prevent permanent evasion from mammalian immune systems, bacterial phage resistance, and non-specific immobilization. Advances in bioinformatics,^30–32 microscopy imaging,^33–35 and genetic engineering^36–38 have created new opportunities for modifying phage structural features to overcome some limitations of current phage-based biotechnologies. Capsids are highly ordered proteinaceous structures utilized by phages to protect their genomes. Phages with the most frequently identified capsid shapes, filamentous and icosahedral,³⁹ have been modified using genetic and chemical methods to functionalize phages with an array of organic and inorganic materials. For phage therapy applications, capsid modifications with polyethylene glycol have increased phages’ bloodstream half-life in vivo.⁴⁰ To generate phage activated materials, site-specific capsid modifications have facilitated binding interactions with silica, cellulose, and magnetic particles to improve immobilized phages’ orientation, stability, and density.^41–43 The potential of phage-based biotechnologies have expanded through the use of non-native proteins displayed on capsids to include applications of cell differentiation scaffolds,^44–48 targeted imaging platforms,^49–51 vaccines,^52–54 and nanocarriers.^55–57 This review adds to the excellent reports of Büning⁵⁸ and Xu⁵⁹ which largely focus on adenovirus capsid modification, where here advances in bacteriophage capsid engineering are highlighted. These genetic and chemical methods for modifying phage capsids can be applied to create a variety of phage-based biotechnologies including low-cost diagnostics, gene therapies, cell differentiation scaffolds, or vaccines.

2. CAPSIDS OVERVIEW

Capsids encapsulate the tightly packed nucleic acids of the phage. Icosahedral capsid geometries having 20 triangular faces are the most frequently observed phage capsid structures.³⁹ These capsids are typically composed of high numbers of repeating structures of a relatively few protein types. The triangulation number of the icosahedral capsid’s central body and two caps can be used to further classify the capsid as the uniform isometric shape or the elongated prolate form.⁶⁰ These capsids vary in complexity and stability depending on the capsid subunits’ composition (pentameric or hexameric), copy number, and organization.^61–63 Most icosahedral phages assemble utilizing scaffolding domains or proteins to aid in proper formation of a stable procapsid shell intermediate which are subsequently removed from the mature capsid before or during genome packaging.^64–66 During the capsid maturation process, some phage capsids expand to reveal binding sites for decoration proteins⁶⁷ that provide a fitness advantage such as increased capsid stability.^68–71 Mature capsids range in size from 43 to 160 nm in diameter.^72,73 The spherical-like shape of these capsids has been utilized for protected delivery of therapeutic and imaging agents through internal modification of empty capsids.^55,74–78

Filamentous phages from the Inoviridae family have a rod like capsid shape that can be 800-2,000 nm long and 6.5-7.5nm in diameter.⁷⁹ Filamentous capsids have a helical array which can be further identified as class I or class II based on having five-fold or one-fold rotational symmetry.^80,81 These capsids are constructed by anchoring all five structural proteins to the inner membrane of the bacteria host and assembling the capsid around the genome as it is translocated into the inner membrane.⁸² The length of the capsid is dictated by the genome size and can be changed by adjusting the genome length.^83,84 High density display of foreign molecules has been achieved by modifying the major capsid protein of filamentous phages that is present in thousands of copies in the capsid.^85–87

Some phages utilize lipid envelopes derived from the phospholipids of the bacteria host to aid in protecting their genome.^88–90 One such phage family which surround their capsids with an external lipid envelope is the Cystoviridae.⁹¹ Corticoviridae, Sphaerolipoviridae, and Tectiviridae phages contain a lipid membrane surrounding the genome on the inside of their capsid.^92–94 To date, the Plasmaviridae phage MVL2 is the only phage to have a lipid membrane as the sole protection surrounding its genome.⁹⁵ The structural characteristics of lipid-containing phages have enabled them to be used as surrogates for enveloped pathogenic human viruses including Ebola virus, severe acute respiratory syndrome (SARS) coronavirus, and human immunodeficiency virus (HIV).^96–100

Phages of all capsid types can be noncovalently immobilized to charged materials via electrostatic adsorption. Important to note is that solution conditions can affect bioconjugation by simple charge-charge interactions, with pH and salt concentration affecting the charge state of capsid protein amino acid residues.¹⁰¹ Charged amino acid residues on phage capsids can be utilized to direct the orientation of phages during bioconjugation, with reported electrostatic adsorption onto modified silica,^102,103 cellulose,^104–106 and gold^103,107 to create bioactive materials. The charge of M13 phages has been exploited to create highly ordered phage monolayer films via electrostatic layer by layer technique.¹⁰⁸ To enhance phage adsorption capabilities, commonly used materials can be modified with charged functional groups including amines, carboxylic acids, and glycols to facilitate adhesion.^{102,105,109,110} This immobilization method is simple, but the nonspecific nature can result in structural features crucial for infection to be interfered with during immobilization. An electric field can be incorporated into the process to properly orient phages during immobilization. This strategy has previously been used to increase the effect of charge during immobilization to direct bioconjugation of T4 phages by their negatively charged capsids during phage-based biosensor construction.^107,111

3. GENETIC MODIFICATIONS

There are numerous genetic engineering approaches that can be used to modify phage capsid genes. Some methods rely on incorporation of the foreign DNA sequence into the phage genome by flanking it with DNA sequences identical to the desired insertion site in the genome. This “Donor DNA” construct creates a template that can seamlessly incorporate the foreign sequence into the phage genome via homologous recombination (Fig 1A). Recombination rates of phages with plasmid vectors containing a Donor DNA construct occur at very low frequencies ranging from 10⁻¹⁰ to 10⁻⁴, ^112–114 but increasing the length of the flanking DNA sequences identical to the insertion site can be used to increase recombination frequency.¹¹⁵ Bacteria hosts used for genetically modified phage synthesis can be altered to increase recombinant phage generation. ¹¹⁶ The in vivo recombineering method utilizes an Escherichia coli strain containing an extra set of temperature inducible recombination genes to help facilitate homologous recombination.^117,118 This E. coli strain is infected with a wild type phage to deliver the phage genome into the cell, heated to induce recombination gene expression, and introduced with a single or double stranded Donor DNA construct that is delivered into the cell via electroporation, typically yielding 0.5-2% phage recombinants.¹¹⁸ Bacteriophage recombineering of electroporated DNA (BRED) method utilizes a bacteria strain containing a plasmid encoding recombination genes that promote high levels of recombination.¹¹⁶ A wild type phage genome and a double stranded Donor DNA construct are simultaneously delivered into the bacteria cell via electroporation which typically results in recombinant phage generation at a frequency of 10-15%.¹¹⁶ Bacteria can be equipped with clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) to form a CRISPR/Cas system within the cell, which were originally derived from a bacteria defense mechanism able to identify and cleave foreign nucleic acids that invade the bacteria cell.¹¹⁹ This system has been adapted into a genetic engineering tool that can be programmed to cleave a targeted gene at a specific location.¹²⁰ The genes required for the CRISPR/Cas system to function can be incorporated directly into the bacteria’s genome or encoded on a plasmid that is maintained in the cell. CRISPR/Cas systems have been programmed to cleave wild type phage genes resulting in a selection of recombinant phages from a mixed pool.¹²¹ CIRSPR/Cas systems can also be used in combination with a plasmid containing a Donor DNA construct to improve recombination rates with the cleaved gene, due to DNA repair mechanisms activated when there is a double stranded DNA break. This method has resulted in a rate of recombination of 99% for some T4 phage genes.³⁶

Figure 1.

Genetic approaches for modified phage capsid engineering, display, and screening. (A) The main approaches for engineering phage genes are highlighted. In homologous recombination, a plasmid containing a donor DNA insert flanked by regions of homology to the desired insert site can be used to facilitate donor DNA insertion into a wild type phage genome to generate a recombinant phage. For in vitro assembly, phage genome fragments synthesized with overlapping ends can be stitched together with the aid of enzymes to construct a recombinant phage genome outside of the bacteria cell. In CRISPR/Cas9 systems, an enzyme-RNA complex can be used to specifically cleave a target sequence in the phage genome to increase the rate of recombination with donor DNA or select out wild type phages. (B) In phage display, genetic engineering is used to fuse an amino acid, peptide, or protein sequence to phage capsid gene resulting in display of the foreign gene product on the phage capsid. In affinity screening, repeated rounds of selection can be used to identify recombinant capsid sequences with strong affinity to the desired target from phage display libraries.

Genetic engineering approaches relying on a bacterial host can be limited by the destruction rate of the host during the lytic phage infection cycle that results in bacteria cell lysis. To overcome the limited rection time, phage genome recombination can be performed in a yeast cell intermediate, Saccharomyces cerevisiae, before insertion of the phage genome into the bacteria host via electroporation.¹²² DNA sequences overlapping with a shuttle plasmid compatible in yeast and bacteria are incorporated into the ends of the phage genome to allow for genome transport between organisms using the shuttle plasmid¹²³ This method has been successful in engineering multiple phages from the T7-family.¹²² In vitro phage genome assembly is another recombination method that is performed independent of the bacteria host. DNA fragments are synthesized with overlapping ends via polymerase chain reactions (PCR) that can hybridize to form the desired recombinant genome sequence with the aid of enzymes.^124,125 The recombinant genome is then introduced into the bacteria host via electroporation to allow for generation of a functional phage This approach has been used to assemble full genomes of a ϕX174 phage and a T7 reporter phage.^126,127 However this method has a higher chance of introducing mutations through PCR errors and can be challenging when transforming large phage genomes into the bacterial host.¹²⁶ Gram positive bacteria’s thick cell wall make it challenging to deliver DNA into the cell via electroporation, limiting in vitro genome assembly applications for phages with gram positive bacteria hosts. To overcome this limitation, cell wall deficient (L-form) bacteria cells can be used to uptake large molecules of DNA.¹²⁸ This approach has been used with L-form Listeria monocytogenes cells to uptake genomes of Listeria, Bacillus, and Staphylococcus phages via PEG mediated transfection.¹²⁹

3.1. PHAGE DISPLAY

The well-established “Phage Display” method is used in combination with a genetic engineering method to construct a modified phage capsid.^130–132 Phage display involves genetically fusing a DNA sequence encoding for a foreign amino acid, peptide, or protein to a phage capsid gene that results in a phage displaying the recombinant capsid protein (Figure 1B). Modified capsid genes can be incorporated into the phage genome or exogenously expressed from a plasmid vector. Synthesis of genetically modified capsid proteins occurs in one production step within the bacteria cell. A mixture of modified and unmodified capsid proteins can be synthesized by incorporating a wild type and a modified capsid gene into the system to improve modified capsid stability.^133–135 Inducible promoters can be used to alter the percent of modified proteins displayed in mixed display systems.^136,137. This method can be used on multiple capsid genes to create a multifunctional phage.^86,138 Phage display is time intensive upfront for proper design and development of the initial modified plasmids and genomes, but once established aliquots can be readily propagated and maintained for use in subsequent production batches.

Phage display libraries composed of a multitude of uniquely modified phage capsids can be subjected to bio-panning for identification of modified phages that best suit the desired application. Phage genotype and phenotype are linked, enabling traceback to reproduce the high preforming strains. Libraries can be randomly generated or specifically altered to elicit specific modification characteristics on the capsid. For example, cyclic peptide modified capsid libraries can be generated by intentionally incorporating cysteine residues to facilitate disulfide bond formation in all strains.^139,140 Bicyclic peptide libraries can be generated by genetically incorporating three cystines into the capsid gene followed reaction with a reagent containing three thiol-reactive groups after phage capsid assembly.^141–145 Forming peptides with nonreducible bonds has been achieved in libraries by utilizing enzymes to catalyze peptide crosslinking¹⁴⁶ or non-canonical amino acids that can undergo specific covalent reactions.^147,148 In vitro bio-panning assays have been used to identify modified phages with affinity to a variety of biological and inorganic substances including single-crystal semiconductors,¹⁴⁹ silica,¹⁵⁰ streptavidin,¹⁵¹ cellulose,¹⁵² and cell surface receptors.^153,154 In vivo bio-panning assays have been developed to identify modified phages that hone specific tissues in mouse^155–157 and human¹⁵⁸ models. Co-display systems of enzymes and substrates have been developed to identity catalytically active modified capsids during bio-panning.^159–165

3.2. AMINO ACIDS

Single amino acids can be added or substituted in capsids to modify the number of functional groups present in side chains capable of reacting in downstream chemical modification steps (Figure 2A).^166–168 By substituting a single amino acid for lysine in the major coat protein of M13 phage, Tridgett et al. were able to conjugate an additional 520 exogenous molecules to the phage via amine conjugation compared to the wild type.¹⁶⁹ In another study, a M13 phage displayed protein was modified to leave only one reactive cystine to increase site specificity of the subsequent thiol conjugation.¹⁷⁰ A similar method can be used to incorporate single unnatural amino acids that contain functional groups uncommon in nature to expand reaction capabilities.^171–175 Rare, nonsense, quadruplet, or reassigned amino acid codons can be added to a gene’s sequence to create a site for unnatural amino acid incorporation by the corresponding unnatural amino acid specific t-RNA and t-RNA synthetase.¹⁷⁶ Unnatural amino acid modification reduces the potential for reacting unintended parts of the capsid but requires a more complex synthesis process that typically results in significantly lower yields compared to wild type protein synthesis.^177,178 This method was used in a phage-based biosensor to modify capsids of T4 phages for immobilization to ensure tail fibers crucial for infection were not altered.¹⁷¹

Figure 2.

Genetic modifications to phage capsids. (A) Single amino acids in phage capsids can be substituted to alter the number and type of functional groups accessible for downstream chemical modification. (B) Peptide motifs recognized by specific enzymes can be incorporated into phage capsids for downstream enzymatic modification or controlled release of contents. (C) Recombinant capsid decoration proteins can be synthesized separately from the phage and assembled to the capsid in vitro, allowing for large complex proteins to be displayed.

3.3. PEPTIDE MOTIFS

Peptide motifs are short structural regions conserved among different proteins that can often be linked to a particular biological function.¹⁷⁹ Peptide motifs that can recognized by enzymes can be displayed on the capsid to create a specific site for enzymatic modification after phage capsid assembly (Figure 2B). Protease cleavage sites have been added to T4 capsid gene constructs to allow for an affinity peptide to be displayed for chromatography purification of phage preparations that can be subsequently removed.¹⁸⁰ M13 and P22 phage nanocarriers containing protease cleavage sites have been used to facilitate enzyme driven release of contents.^49,181 Biotin ligase enzymes can utilize ATP to conjugate biotin to biotin carboxyl carrier protein (BCCP) via an amide linkage to one of BCCP’s lysine residues. The components of this system can be used to biotinylate BCCP displaying phages in vitro.¹⁸² Biotinylating can also occur in vivo during normal phage propagation, if a biotin ligase enzyme gene is present in the bacteria host or genetically incorporated into the phage or bacteria host.^183,184 BCCPs have been incorporated into T4, M13, and T7 phages to allow for site specific biotin addition.^57,185–188 Several commercially available biotin and streptavidin functionalized materials offer a diverse array of applications for BCCP tagged phages.

Sortase enzymes (those which modify surface proteins) can be incorporated into phage display systems to catalyze covalent bond formation between the N terminus of a substrate peptide or peptide conjugate to the C terminus of a cleaved sortase recognition motif.¹⁸⁹ Commonly used sortase A transpeptidation systems rely on a five amino acid long recognition motif and two-five amino acid long substrates.^190–192 This method has been utilized to conjugate green fluorescent proteins and influenza antigens to P22 capsids.¹⁸⁹ Sortase motifs derived from different organisms have been used in the same system on different M13 capsid proteins to create multifunctional modified phage capsids.^193,194 After reacting for 3 hours, 56-74% of the M13 minor capsid proteins were attached with green fluorescent protein or biotin via sortase ligation.¹⁹³ Sortase catalyzed modification is advantageous for facilitating attachment of large complex proteins that may disrupt stable capsid assembly if fused directly to the phage capsid gene. This system is limited by the inefficiency of ligation by the sortase enzymes used which can be compensated for by using longer reaction times.¹⁹⁵

3.4. DECORATION PROTEINS

Decoration proteins typically incorporated into icosahedral capsids after procapsid shell formation can assemble in vitro to mature capsids of strains where the gene for the decoration protein has been knocked out (Figure 2C).¹⁹⁶ This method allows for more purification and characterization of the modified proteins before incorporating them into the capsid to ensure they are in the correct form. It also allows for the displaying complex proteins that typically cannot be efficiently synthesized in the phage’s bacteria host due to the protein’s complexity or toxicity. Antigen constructs over twice the size of the decoration protein it is fused to have been successfully displayed on all capsid binding sites of T4 phage using this method.¹⁹⁷ Multiple antigen fusions to the same T4 decoration protein have been synthesized separately then mixed in equal molar ratios before in vitro binding to allow for mixed display of antigens on a single phage capsid.¹⁹⁸ In vitro assembly has been demonstrated in T4 phage with displayed antigens of swine fever virus,⁵⁴ human immunodeficiency virus,⁵³ foot and mouth disease virus,⁵² Bacillus anthracis,¹⁹⁷ and Neisseria meningitidis¹⁹⁹ for initial vaccine development. Decoration protein homologs with conserved capsid binding domains have been bound to closely related phages to offer improved solubility or quantification capability compared to the native decoration proteins.^200–202

4. CHEMICAL MODIFICATIONS

Another method for manipulating phages’ structure and function is through chemical modification. Amino acids in the proteinaceou6s phage capsids offer a variety of reactive functional groups available for bioconjugation including carboxylic acids, amines, phenols, and thiols (Figure 3). The maximum degree of modification is determined by the number of reactive groups present that are sterically accessible for modification, their pKa, and solution conditions. The plethora of nucleophilic functional groups present creates potential for several amino acids to participate in chemical reactions. However, this can result in mixed reaction products from undesired side reactions even when conditions are optimized to favor a particular group.^203,204 Unintentional modification of residues that are crucial for proper structural feature function can decrease phage infectivity.⁴⁰ Low abundance amino acids such as cysteine or unnatural amino acids can be targeted for improved control over the site of modification.²⁰⁵ Phospholipids in lipid enveloped phages provide another target for modification. Human enveloped viruses have been modified through propagating in a host engineered to metabolically incorporate chemically functionalized components into their cell membranes.^206–208 This hybrid method provides a potential pathway for enveloped phage modification in the future.

Figure 3.

Chemical modifications of filamentous phage capsids. Functional groups present on amino acids or unnatural amino acids can be utilized to add desired conjugates to phage capsids in a semi-selective manner. Though depicted on a filamentous phage capsid, these modification chemistries can be applied to other phage capsid architectures.

4.1. AMINE GROUPS

Amine groups present at the N-terminus of all proteins and on lysine side chains are common targets for chemical modification. Reaction pH can be used to drive the reaction towards the α-amino group at the N-terminus (pKa ~8) or ε-amino group of lysine (pKa ~10) but still usually results in mixed modification.^209,210 Models have been created to help predict the molar ratio of NHS (N-hydroxysuccinimide) ester reagent to target protein to achieve the desired degree of modification.^211,212 NHS ester reagents are frequently used to form stable amide linkages with amines in one-step reactions. Several NHS ester conjugates are commercially available and stable under dry conditions. NHS esters in homo- and hetero-bifunctional reagents have been used to crosslink T4 phages to gold sensors and MS2 capsids to cell penetrating peptides.^168,213 NHS esters modified with a negatively charged sulfonate group can be used to increase the reagents solubility in water and reduce the need for organic solvents in reactions.²¹⁴ NHS^215–220 and Sulfo-NHS^168,221,222 esters have been used to modify phages from a variety of families including T7, M13, T4, A511, Felix-O1, SJ2, and MS2. Nonionic polyethylene glycol (PEG) linker arms have also been incorporated into NHS ester conjugates used for M13, fd, and G1 phage modifications to improve solubility and biocompatibilty.^223–225 Basic conditions required for amine deprotonation can be problematic with these reactions, because NHS esters are highly susceptible to base hydrolysis.^226–228 Tetrafluorophenyl (TFP) esters are another water soluble reagent able to form amide bonds with amines but are more stable in basic pH conditions and more hydrophobic.²²⁹ Isocyanates and isothiocyanates can react with amines to form ureas and thioureas. Isothiocyanates are used more frequently for bioconjugations than isocyanates, because they are more stable in storage.^230,231 TFP esters^232,233 and isothiocyanates^216,234 have been used to modify T7, MS2, and M13 phages with fluorophores. For increased specificity, a two-step reaction can be used to modify only α-amino groups at the N-terminus of proteins under mild conditions. First the phage is incubated with pyridoxal 5’-phosphate (PLP) to undergo a transamination reaction that swaps the N-terminal amine for a ketone.²³⁵ This ketone can undergo an oxime reaction with an aminooxy-functionalized substituent, which has been used for generation of fd phage biosensors.^236,237 N-terminal alanine residues with proximal lysine residues have shown to increase the efficiency of this bioconjugation reaction.²³⁸ This method has been used for high density attachment of imaging agents and polymers to fd phage capsids.⁵¹

4.2. CARBOXYLATE GROUPS

Carboxylate groups are found at the C-terminus of proteins and on aspartate and glutamate side chains. Carboxylates have a low reactivity in water so they are commonly activated with a carbodiimide crosslinker like the water soluble 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) or water insoluble Dicyclohexylcarbodiimide (DCC) to form a reactive O-acylisourea intermediate.²³⁹ Activated carboxylates can then be reacted with primary amines in mildly acidic pH conditions to form stable amide bonds that leave no trace of the carbodiimide used to facilitate the bond formation.²⁴⁰ EDC coupling reactions have been used to conjugate reactive moieties and chloramphenicol to filamentous phages as shown in Table 1.^241–243 NHS esters can be added to these reactions to generate a more stable NHS ester intermediate that can undergo reaction with primary amines under physiological pH.²⁴⁴ This method has been used to attach fluorphores²²², magnetic beads,²²⁵ folic acid,²⁴⁵ and drugs^49,246 to M13 phage capsids. Glutaric anhydride has been conjugated to primary amines on M13 phages to increase the number of carboxylate groups accessible for polymer immobilization in downstream reactions.²⁴⁷

Table 1.

Modified Phage Capsids Summary

GENETIC
Protease Cleavage Site Addition	AcTEV protease mediated affinity tag removal180 Cathepsin B mediated release of chemotherapeutic49 Thrombin mediated release of fluorescent protein181
Biotin Carboxyl Carrier Protein Addition	Horseradish peroxidase conjugation57 Immobilization on gold surfaces185 Antibody conjugation188 Quantum dot conjugation186 Immobilization on magnetic beads187
Sortase Recognition Motif Addition	Green fluorescent protein conjugation189,193 Influenza antigen conjugation189 Biotin conjugation193
In Vitro Display	Anthrax protective antigen conjugation197 Anthrax protective antigen, lethal factor, and edema factor conjugation198 Neisseria meningitidis PorA peptide conjugation199 Foot and mouth disease virus capsid precursor polyprotein or proteinase peptide conjugation52 Human immunodeficiency virus antigens gp24, Nef, and gp41 conjugation53 Classical swine fever virus primary antigen and major antigenic determinant cluster conjugation54
CHEMICAL
NHS Ester	PEG conjugation40 Fluorescent dye conjugation199,201 Biotin or PEG-Biotin conjugation200,203,206,207
NHS Ester; Oxime Formation	Magnetic resonance contrast agent conjugation74
NHS Ester; Metal Binding	Gold nanorod conjugation220
Sulfo-NHS Ester	Biotin conjugation221
TFP Ester	Fluorescent dye conjugation232,233
Isothiocyanate	Fluorescent dye conjugation216,234
EDC + NHS Ester Coupling	Immobilization to superparamagnetic particles225
DCC + NHS Ester Coupling	Chloramphenicol conjugation246
EDC + Sulfo-NHS Ester Coupling	Doxorubicin conjugation49 Fluorophore conjugation222 Folic acid conjugation245
EDC Coupling	Cysteamine conjugation241 Chloramphenicol conjugation242,243
Diazonium; Copper Catalyzed Azide-Alkyne Cycloaddition	Biotin or folate conjugation222
Diazonium; Oxime Formation	Magnetic resonance contrast agent conjugation74
PLP Transamination; Oxime Formation	PEG conjugation51 Fluorescent dye conjugation51 Xe binding molecule conjugation237
Glutaric Anhydride Addition	Polymer conjugation247
Metal Bonding	Gold nanoparticle conjugation241,257
Glutaraldehyde Crosslinking	Immobilization on amino acid functionalized gold surfaces110 Immobilization on amine functionalized magnetic microspheres264
Sodium Periodate Oxidation; Oxime Formation	Biotin conjugation267 Mannose conjugation267
2-Amino Benzamidoxime	Biotin conjugation268
HYBRID*
Amino Acid Substitution; Maleimide	Biotin or PEG-Biotin conjugation166,170 Fluorescent dye conjugation76,167,169,253
Amino Acid Substitution; Sulfo-NHS Ester; Maleimide	Crosslinking cell penetrating peptides to phage capsids168
Amino Acid Substitution; Isothiocyanate	Fluorescent dye conjugation169
Sortase Recognition Motif Addition; Maleimide	Fluorescent dye conjugation194 DNA conjugation194
Unnatural Amino Acid Addition; Selenide-Sulfide Crosslinking	Biotin conjugation269,270
Unnatural Amino Acid Addition; Copper Catalyzed Azide- Alkyne Cycloaddition	Gold nanoparticle conjugation175 Fluorescent dye conjugation172 Magnetic bead conjugation171
Unnatural Amino Acid Addition; Sodium Periodate Oxidation	Cell penetrating peptide conjugation276 Antibody conjugation277

Left column is the modification method used. Right column is the application.

^*Hybrid includes genetic and chemical modification methods.

4.3. THIOL GROUPS

Thiol groups in cysteine side chains are the most reactive nucleophiles present in proteins.²⁴⁸ Under oxidizing conditions, thiols from two cystines can form a disulfide bond. Thiols in cystine disulfide bonds can be liberated using reducing agents to increase the number of thiols accessible for modification, but this can cause protein destabilization or reversion back to disulfides.^249,250 Maleimides react with thiols under mild pH and temperature conditions to form stable thioether bonds.²⁵¹ Maleimides can also react with amines but their reaction with sulfhydryl groups occurs 1000-fold faster at pH 7 therefore is highly favored.²⁵² Maleimide conjugates functionalized for fluorescence,^167,241,253 water solubility,¹⁶⁶ neomycin²⁴² and improved stability^168,170 have been used to modify P22, MS2, M13, and fd phages. Thiol groups can also readily form dipolar bonds with metal ions and materials.^254–256 These bonds are not as strong as traditional covalent bonds, but allow even disulfide bonds to form bonds with metals.²⁵² Based on this principle, cysteine residues have been incorporated into fd phage capsids to improve binding to gold materials.²⁵⁷ N-succinimidyl-S-acetylthiopropionate (SATP) has been chemically conjugated to M13 phages to increase the number of thiol groups capable of bonding with gold nanorods.²²⁰

4.4. PHENOL GROUPS

Phenol groups present on tyrosine and histidine residues can be modified using diazonium compounds to form diazo linked conjugates. Adjusting the reaction pH to 7 favors reaction with the histidine imidazole group, where higher pH’s favor reaction with the tyrosine phenol group.²⁵⁸ This strategy has been utilized to facilitate conjugation of imaging agents to M13 and MS2 phage capsids via tyrosine residues^74,222

4.5. ALDEHYDE CROSS LINKERS

Glutaraldehyde is a dialdehyde that is frequently used as a crosslinking agent to form chemically and thermally stable crosslinks of biological materials.^259,260 Glutaraldehyde is present in many forms in solution leading to the precise mechanism and primary reactive species not being fully understood or agreed upon in the literature.²⁶¹ Glutaraldehyde can react with several nucleophilic functional groups present in proteins including amines, thiols, phenols, and imidazole, but the ε-amino group of lysine was found to be the most reactive.^262,263 This method has been used to facilitate crosslinking phages to amine functionalized magnetic microspheres and gold surfaces.^110,264 N-terminal serine and threonine residues contain a β-amino alcohol motif that can specifically undergo oxidative cleavage by sodium periodate to generate an aldehyde handle.²⁶⁵ This handle can be subjected to a second reaction to add the desired conjugate. Oxime reactions have been used in aldehyde displaying phages to attach aminooxy conjugates. Oxime reactions can take several hours at neutral pH but can be accelerated using acidic conditions (pH 4.5) and an aniline catalyst.²⁶⁶ This two-step method was used in a one-pot reaction to add an aminooxy-functionalized glycan to M13 phages through an oxime condensation reaction in less than 1.5 hours.²⁶⁷ In another study, an aldehyde conjugated to a NHS ester was used to attach aldehydes to amines on the exterior of MS2 phage capsids, followed by an 8 hour oxime condensation reaction at pH 6.5 to attach the desired contrast agent.⁷⁴ In a similar reaction, phage displayed aldehydes with 2-amino benzamidoxime derivatives have been used to conjugate materials to M13 phages in one hour resulting in bonds more resilient to hydrolysis than oximes.²⁶⁸

4.6. UNNATURAL AMINO ACIDS

Amino acids that are not encoded for in the natural genetic code of organisms can be incorporated into phage capsid protein constructs to display unique functional groups to generate sites on the capsid for selective chemical modification. Selenocysteine is an unnatural amino acid cysteine analog containing selenium in place of sulfur that has been incorporated into M13 phages for biotin conjugation.^269,270 This strong nucleophile reacts fast and at a low pH which can help prevent undesired side reactions that occur at a higher pH.²⁷¹ Multiple uncommon or unnatural amino acids have been synthesized to contain azide or alkyne groups capable of undergoing a copper catalyzed azide-alkyne cycloaddition click chemistry reaction.^272–274 This fast, highly specific reaction occurs among functional groups that are not common in biological substances, making it a good candidate for site specific modification with limited potential for unwanted side reactions.²⁷⁵ Azide containing unnatural amino acid displaying M13 phages have facilitated conjugation to alkyne functionalized fluorophores and gold particles.^172,175 A tyrosine unnatural amino derivative, p-aminophenylalanine, can undergo sodium periodate mediated oxidative coupling for phage capsid bioconjugation. This method has been used to modify MS2 phage like particles and shown selective even in the presence of tyrosine.^76,276,277

5. OPPORTUNITIES AND CHALLENGES

The modification approaches described in this review highlight advances made to functionalize phages for a diverse array of applications. These strategies can be used alone or in combination depending on the phage’s characteristics and desired end modification. Adsorption to materials through electrostatic interactions offers the simplest method for functionalizing phages but lacks specificity and permanence. Genetic engineering can be used to fuse DNA sequences encoding for amino acids, peptides, or proteins to phage capsid genes through the well-established “Phage Display” method. These recombinant capsid proteins create additional sites for capsid modification by enzymes or chemicals. To accommodate large complex foreign protein fusions, recombinant capsid decoration proteins can be assembled in vitro to phages with accessible binding sites. Chemical modifications can also be used to target functional groups on amino acids of phage capsids for modification. Chemical reactions have potential for undesired side reactions to occur, but pH and temperature conditions can be adjusted to drive the reaction towards a particular functional group to mitigate side reactions. Protective groups can also be utilized during multistep reactions to improve selectivity of the reaction.

The plethora of genetic and chemical phage modification strategies have expanded the possibilities of phage-based biotechnologies. Hybrid methods utilizing genetic engineering to incorporate a low abundance or unnatural amino acid residue that can undergo a specific chemical reaction downstream appear to be the most promising for specific capsid modification. Current site-specific chemical methods are time-intensive and require multiple steps leaving much room for improvement to streamline the phage engineering process. To date, icosahedral and filamentous phage capsids have been the most frequently modified with no reported lipid modification of enveloped phages. Future studies adapting human enveloped virus modification methods to enveloped phages could tap into an underutilized capsid feature. Decoration proteins have been substituted from closely related phages to improve recombinant phage solubility. Investigating substitutions of other closely related phage capsid proteins could be a promising approach to alter phage capsid properties. Methods described in this review for capsid protein modification can be adapted to modify other phage proteins. For example, phage tail fibers that act as receptors for binding to bacteria hosts can be modified to alter a phage’s bacteria host range. Overall, increased characterization of different phage capsids at the genomic and structural level will help expand the already promising opportunities of phage capsid engineering highlighted in this review.

ABBREVIATIONS

BCCP

biotin carboxyl carrier protein

BRED

bacteriophage recombineering of electroporated DNA

CRISPR

clustered regularly interspaced palindromic repeats

Cas

CRISPR-associated proteins

DCC

Dicyclohexylcarbodiimide

EDC

1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride

HIV

human immunodeficiency virus

NHS

N-hydroxysuccinimide

PCR

polymerase chain reaction

PEG

polyethylene glycol

PLP

pyridoxal 5’-phosphate

SARS

severe acute respiratory syndrome

SATP

N-succinimidyl-S-acetylthiopropionate

TFP

tetrafluorophenyl