Phage-Derived Antibacterials: Harnessing the Simplicity, Plasticity, and Diversity of Phages

Abstract

Despite the successful use of antibacterials, the emergence of multidrug-resistant bacteria has become a serious threat to global healthcare. In this era of antibacterial crisis, bacteriophages (phages) are being explored as an antibacterial treatment option since they possess a number of advantages over conventional antibacterials, especially in terms of specificity and biosafety; phages specifically lyse target bacteria while not affecting normal and/or beneficial bacteria and display little or no toxicity in that they are mainly composed of proteins and nucleic acids, which consequently significantly reduces the time and cost involved in antibacterial development. However, these benefits also create potential issues regarding antibacterial spectra and host immunity; the antibacterial spectra being very narrow when compared to those of chemicals, with the phage materials making it possible to trigger host immune responses, which ultimately disarm antibacterial efficacy upon successive treatments. In addition, phages play a major role in horizontal gene transfer between bacterial populations, which poses serious concerns for the potential of disastrous consequences regarding antibiotic resistance. Fortunately, however, recent advancements in synthetic biology tools and the speedy development of phage genome resources have allowed for research on methods to circumvent the potentially disadvantageous aspects of phages. These novel developments empower research which goes far beyond traditional phage therapy approaches, opening up a new chapter for phage applications with new antibacterial platforms. Herein, we not only highlight the most recent synthetic phage engineering and phage product engineering studies, but also discuss a new proof-of-concept for phage-inspired antibacterial design based on the studies undertaken by our group.

Keywords: engineering; life cycle; lysin; phage; pyocin.

Figure 1

Synthetic phage engineering strategies. Major strategies for modifying the target recognition site (tail), phage head (hexagonal), or genome (tangled lines) are depicted. The bacterial host spectrum of a phage can be expanded or redirected by tail shuffling (orange), while mammalian host immune responses can be reduced or minimized by insertional inactivation of the endolysin gene (red triangle) or capsid modification (green circles). Heterologous effector genes (yellow rectangle) can also be installed on the phage genome to maximize the antibacterial efficacy.

Figure 2

Endolysin engineering strategy. The modular structures of endolysins are shown; catalytic domains (enzymatically active domain (EAD)) of endolysin A and endolysin B (green and white rectangles) are originally connected to their binding domains (cell wall-binding domain (CDB)) (grey and red rectangles) via cognate linkers (straight and wavy lines). An engineered chimeric endolysin can be created by domain shuffling using the endolysin A EAD and the endolysin B CBD with an appropriate linker.

Figure 3

Pyocin engineering strategy and mode of action. The target of an R-type pyocin can be shifted from Pseudomonas aeruginosa to Escherichia coli or Clostridium difficile by tail shuffling. The tail fibers of either (A) E. coli phage or (B) C. difficile phage are fused to the R-pyocin, resulting in modified R-pyocins that can bind to respective target bacterial membranes. Once the modified R-pyocins reach each hosts’ membranes, sheath contraction induces core tube penetration and disrupts the membrane potential (red bars).

Figure 4

Work flow of phage-inspired antibacterial design. Once phages with strong antibacterial activity are identified, their whole genomes are sequenced and analyzed for open reading frame (ORF) selection. The selected ORFs are expressed in target bacterial hosts to screen for antibacterial activity based on growth or virulence inhibition. Then, the gene product of an ORF hit (dark green) is exploited to fish out the host target based on protein–protein interactions. The discovered host target, the phage-inspired bacterial target, serves as a molecular scaffold for new drug screening. Finally, chemicals or peptides that bind to the host target can be isolated and further developed as phage-inspired antibacterials.