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.

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.

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.

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.