Phage Engineering Research Articles

Assessing the transcriptional landscape of Pseudomonas phage 201ϕ2-1: Uncovering the small regulatory details of a giant phage.

The transcriptional architecture of phages can deepen our understanding of the phage-host infection process and can be of key importance for phage engineering and biotechnological applications. Here, we applied ONT-cappable-sequencing, a long-read RNA-sequencing technique, to study the regulatory mechanisms of Pseudomonas infecting giant phage 201ϕ2-1. We identified 67 promoters and 132 terminators that together represent 92 transcriptional units. A full comparison of these data to the transcriptome of model Pseudomonas phage ϕKZ confirmed that the transcriptional programs of these prototypes of the Serwervirus and Phikzvirus genera are largely conserved, despite some subtle regulatory differences. Evidence supporting these shared mechanisms include the identification of highly similar sequence motifs for regulatory elements in both phages and the conservation of regulatory elements loci relative to homologous genes in each phage. Moreover, we discovered a sRNA in 201ϕ2-1 that is highly conserved among prototype members of different giant phage genera. Sequencing of the 201ϕ2-1 host genome resulted in its reclassification as Pseudomonas atacamensis, a close relative of the important agricultural biocontrol agent Pseudomonas chlororaphis. Finally, we conducted invivo assays of eight 201ϕ2-1 terminators and found them to strongly terminate transcription in P. chlororaphis. Control elements from phage transcriptional programs have a rich history for applications in biotechnology. In these studies, we demonstrate new insight into the transcriptional program of 201ϕ2-1 and demonstrate the potential of its regulatory elements for novel and useful tools for synthetic biology circuitry.

Data-Driven Engineering of Phages with Tunable Capsule Tropism for Klebsiella pneumoniae.

Klebsiella pneumoniae, a major clinical pathogen known for causing severe infections, is attracting heightened attention due to its escalating antibiotic resistance. Phages are emerging as a promising alternative to antibiotics; however, their specificity to particular hosts often restricts their use. In this study, a collection of 114 phages is obtained and subjected to analysis against 238 clinical K. pneumoniae strains, revealing a spectrum of lytic behaviors. A correlation between putative tail protein clusters and lysis patterns leads to the discovery of six receptor-binding protein (RBP) clusters that determine host capsule tropism. Significantly, RBPs with cross-capsular lysis capabilities are identified. The newly-identified RBPs provide a toolbox for customizing phages to target diverse capsular types. Building on the toolbox, the engineered phages with altered RBPs successfully shifted and broadened their host capsule tropism, setting the stage for tunable phage that offer a precise and flexible solution to combat K. pneumoniae infections.

Cell-free synthesis of infective phages from in vitro assembled phage genomes for efficient phage engineering and production of large phage libraries.

Bacteriophages are promising alternatives to traditional antimicrobial treatment of bacterial infections. To further increase the potential of phages, efficient engineering methods are needed. This study investigates an approach to phage engineering based on phage rebooting and compares selected methods of assembly and rebooting of phage genomes. GG assembly of phage genomes and subsequent rebooting by cell-free transcription-translation reactions yielded the most efficient phage engineering and allowed production of a proof-of-concept T7 phage library of 1.8 × 107 phages. We obtained 7.5 × 106 different phages, demonstrating generation of large and diverse libraries suitable for high-throughput screening of mutant phenotypes. Implementing versatile and high-throughput phage engineering methods allows vastly accelerated and improved phage engineering, bringing us closer to applying effective phages to treat infections in the clinic.

Bacteriophage-Based Bioanalysis.

Bacteriophages, which are viral predators of bacteria, have evolved to efficiently recognize, bind, infect, and lyse their host, resulting in the release of tens to hundreds of propagated viruses. These abilities have attracted biosensor developers who have developed new methods to detect bacteria. Recently, several comprehensive reviews have covered many of the advances made regarding the performance of phage-based biosensors. Therefore, in this review, we first describe the landscape of phage-based biosensors and then cover advances in other aspects of phage biology and engineering that can be used to make high-impact contributions to biosensor development. Many of these advances are in fields adjacent to analytical chemistry such as synthetic biology, machine learning, and genetic engineering and will allow those looking to develop phage-based biosensors to start taking alternative approaches, such as a bottom-up design and synthesis of custom phages with the singular task of detecting their host.

Comparing Methods to Genetically Engineer Bacteriophage and Increase Host Range.

Antibacterial resistance is an emerging problem in military medicine. Disruptions to the health care systems in war-torn countries that result from ongoing conflict can potentially exacerbate this problem and increase the risk to U.S. forces in the deployed environment. Therefore, novel therapies are needed to mitigate the impact of these potentially devastating infections on military operations. Bacteriophages are viruses that infect and kill bacteria. They can be delivered as therapeutic agents and offer a promising alternative to traditional antibiotic chemotherapy. There are several potential benefits to their use, including high specificity and comparative ease of use in the field setting. However, the process of engineering phages for military medical applications can be a laborious and time-consuming endeavor. This review examines available techniques and compares their efficacy. This review evaluates the scientific literature on the development and application of four methods of bacteriophage genome engineering and their consideration in the context of military applications. Preffered Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were followed for a systematic review of available literature that met criteria for analysis and inclusion. The research completed for this review article originated from the United States Military Academy's library "Scout" search engine, which compiles results from 254 available databases (including PubMed, Google Scholar, and SciFinder). Particular attention was focused on identifying useful mechanistic insight into the nature of the engineering technique, the ease of use, and the applicability of the technique to countering the problem of antimicrobial resistance in the military setting. A total of 52 studies were identified that met inclusion criteria following PRISMA guidelines. The bioengineering techniques analyzed included homologous recombination (12 articles), in vivo recombineering (9 articles), bacteriophage recombineering of electroporated DNA (7 articles), and the CRISPR-Cas system (10 articles). Rates of success and fidelity varied across each platform, and comparative benefits and drawbacks are considered. Each of the phage engineering techniques addressed herein varies in amount of effort and overall success rate. CRISPR-Cas-facilitated modification of phage genomes presents a highly efficient method that does not require a lengthy purification and screening process. It therefore appears to be the method best suited for military medical applications.

PHEIGES: all-cell-free phage synthesis and selection from engineered genomes

Bacteriophages constitute an invaluable biological reservoir for biotechnology and medicine. The ability to exploit such vast resources is hampered by the lack of methods to rapidly engineer, assemble, package genomes, and select phages. Cell-free transcription-translation (TXTL) offers experimental settings to address such a limitation. Here, we describe PHage Engineering by In vitro Gene Expression and Selection (PHEIGES) using T7 phage genome and Escherichia coli TXTL. Phage genomes are assembled in vitro from PCR-amplified fragments and directly expressed in batch TXTL reactions to produce up to 1011 PFU/ml engineered phages within one day. We further demonstrate a significant genotype-phenotype linkage of phage assembly in bulk TXTL. This enables rapid selection of phages with altered rough lipopolysaccharides specificity from phage genomes incorporating tail fiber mutant libraries. We establish the scalability of PHEIGES by one pot assembly of such mutants with fluorescent gene integration and 10% length-reduced genome.

Refining the transcriptional landscapes for distinct clades of virulent phages infecting Pseudomonas aeruginosa.

The introduction of high-throughput sequencing has resulted in a surge of available bacteriophage genomes, unveiling their tremendous genomic diversity. However, our current understanding of the complex transcriptional mechanisms that dictate their gene expression during infection is limited to a handful of model phages. Here, we applied ONT-cappable-seq to reveal the transcriptional architecture of six different clades of virulent phages infecting Pseudomonas aeruginosa. This long-read microbial transcriptomics approach is tailored to globally map transcription start and termination sites, transcription units, and putative RNA-based regulators on dense phage genomes. Specifically, the full-length transcriptomes of LUZ19, LUZ24, 14-1, YuA, PAK_P3, and giant phage phiKZ during early, middle, and late infection were collectively charted. Beyond pinpointing traditional promoter and terminator elements and transcription units, these transcriptional profiles provide insights in transcriptional attenuation and splicing events and allow straightforward validation of Group I intron activity. In addition, ONT-cappable-seq data can guide genome-wide discovery of novel regulatory element candidates, including noncoding RNAs and riboswitches. This work substantially expands the number of annotated phage-encoded transcriptional elements identified to date, shedding light on the intricate and diverse gene expression regulation mechanisms in Pseudomonas phages, which can ultimately be sourced as tools for biotechnological applications in phage and bacterial engineering.

Use of Cas9 Targeting and Red Recombination for Designer Phage Engineering.

Bacteriophages (phages) are natural antibiotics and biological nanoparticles, whose application is significantly boosted by recent advances of synthetic biology tools. Designer phages are synthetic phages created by genome engineering in a way to increase the benefits or decrease the drawbacks of natural phages. Here we report the development of a straightforward genome engineering method to efficiently obtain engineered phages in a model bacterial pathogen, Pseudomonas aeruginosa. This was achieved by eliminating the wild type phages based on the Streptococcus pyogenes Cas9 (SpCas9) and facilitating the recombinant generation based on the Red recombination system of the coliphage λ (λRed). The producer (PD) cells of P. aeruginosa strain PAO1 was created by miniTn7-based chromosomal integration of the genes for SpCas9 and λRed under an inducible promoter. To validate the efficiency of the recombinant generation, we created the fluorescent phages from a temperate phage MP29. A plasmid bearing the single guide RNA (sgRNA) gene for selectively targeting the wild type gp35 gene and the editing template for tagging the Gp35 with superfolder green fluorescent protein (sfGFP) was introduced into the PD cells by electroporation. We found that the targeting efficiency was affected by the position and number of sgRNA. The fluorescent phage particles were efficiently recovered from the culture of the PD cells expressing dual sgRNA molecules. This protocol can be used to create designer phages in P. aeruginosa for both application and research purposes.

Genetic Engineering and Rebooting of Bacteriophages in L-Form Bacteria.

The rapid increase of circulating, antibiotic-resistant pathogens is a major ongoing global health crisis, and arguably, the end of the "golden age of antibiotics" is looming. This has led to a surge in research and development of alternative antimicrobials, including bacteriophages, to treat such infections (phage therapy). Isolating natural phage variants for the treatment of individual patients is an arduous and time-consuming task. Furthermore, the use of natural phages is frequently hampered by natural limitations, such as moderate in vivo activity, the rapid emergence of resistance, insufficient host range, or the presence of undesirable genetic elements within the phage genome. Targeted genetic editing of wild-type phages (phage engineering) has successfully been employed in the past to mitigate some of these pitfalls and to increase the therapeutic efficacy of the underlying phage variants. Clearly, there is a large potential for the development of novel, marker-less genome-editing methodologies to facilitate the engineering of therapeutic phages. Steady advances in synthetic biology have facilitated the in vitro assembly of modified phage genomes, which can be activated ("rebooted") upon transformation of a suitable host cell. However, this can prove challenging, especially in difficult-to-transform Gram-positive bacteria. In this chapter, we detail the production of cell wall-deficient L-form bacteria and their application to activate synthetic genomes of phages infecting Gram-positive host species.

Genetic Engineering of Therapeutic Phages Using Type III CRISPR-Cas Systems.

The functional characterization of "hypothetical" phage genes is a major bottleneck in basic and applied phage research. To compound this issue, the most suitable phages for therapeutic applications-the strictly lytic variety-are largely recalcitrant to classical genetic techniques due to low recombination rates and lack of selectable markers. Here we describe methods for fast and effective phage engineering that rely upon a Type III-A CRISPR-Cas system. In these methods, the CRISPR-Cas system is used as a powerful counterselection tool to isolate rare phage recombinants.

RNA and Single-Stranded DNA Phages: Unveiling the Promise from the Underexplored World of Viruses

RNA and single-stranded DNA (ssDNA) phages make up an understudied subset of bacteriophages that have been rapidly expanding in the last decade thanks to advancements in metaviromics. Since their discovery, applications of genetic engineering to ssDNA and RNA phages have revealed their immense potential for diverse applications in healthcare and biotechnology. In this review, we explore the past and present applications of this underexplored group of phages, particularly their current usage as therapeutic agents against multidrug-resistant bacteria. We also discuss engineering techniques such as recombinant expression, CRISPR/Cas-based genome editing, and synthetic rebooting of phage-like particles for their role in tailoring phages for disease treatment, imaging, biomaterial development, and delivery systems. Recent breakthroughs in RNA phage engineering techniques are especially highlighted. We conclude with a perspective on challenges and future prospects, emphasizing the untapped diversity of ssDNA and RNA phages and their potential to revolutionize biotechnology and medicine.

Bacteriophages and Their Host Range in Multidrug-Resistant Bacterial Disease Treatment.

The rapid emergence of multidrug-resistant (MDR) bacteria in recent times has prompted the search for new and more potent antibiotics. Bacteriophages (commonly known as phages) are viruses that target and infect their bacterial hosts. As such, they are also a potential alternative to antibiotics. These phages can be broadly categorized into monovalent (with a narrow host range spectrum and specific to a single bacterial genus) and polyvalent (with a broad host range and specific to more than two genera). However, there is still much ambiguity in the use of these terms, with researchers often describing their phages differently. There is considerable research on the use of both narrow- and broad-host range phages in the treatment of infections and diseases caused by MDR bacteria, including tuberculosis, cystic fibrosis, and carbapenem-resistant Enterobacterales (CRE) infectious diseases. From this, it is clear that the host range of these phages plays a vital role in determining the effectiveness of any phage therapy, and this factor is usually analyzed based on the advantages and limitations of different host ranges. There have also been efforts to expand phage host ranges via phage cocktail development, phage engineering and combination therapies, in line with current technological advancements. This literature review aims to provide a more in-depth understanding of the role of phage host ranges in the effectiveness of treating MDR-bacterial diseases, by exploring the following: phage biology, the importance of phages in MDR bacteria diseases treatment, the importance of phage host range and its advantages and limitations, current findings and recent developments, and finally, possible future directions for wide host range phages.

An efficient, scarless, selection-free technology for phage engineering

ABSTRACT Most recently developed phage engineering technologies are based on the CRISPR-Cas system. Here, we present a non-CRISPR-based method for genetically engineering the Escherichia coli phages T5, T7, P1, and λ by adapting the pORTMAGE technology, which was developed for engineering bacterial genomes. The technology comprises E. coli harbouring a plasmid encoding a potent recombinase and a gene transiently silencing a repair system. Oligonucleotides with the desired phage mutation are electroporated into E. coli followed by infection of the target bacteriophage. The high efficiency of this technology, which yields 1–14% of desired recombinants, allows low-throughput screening for the desired mutant. We have demonstrated the use of this technology for single-base substitutions, for deletions of 50–201 bases, for insertions of 20 bases, and for four different phages. The technology may also be readily modified for use across many additional bacterial and phage strains.

The Biotechnological Application of Bacteriophages: What to Do and Where to Go in the Middle of the Post-Antibiotic Era.

Amid the escalating challenges of antibiotic resistance, bacterial infections have emerged as a global threat. Bacteriophages (phages), viral entities capable of selectively infecting bacteria, are gaining momentum as promising alternatives to traditional antibiotics. Their distinctive attributes, including host specificity, inherent self-amplification, and potential synergy with antibiotics, render them compelling candidates. Phage engineering, a burgeoning discipline, involves the strategic modification of bacteriophages to enhance their therapeutic potential and broaden their applications. The integration of CRISPR-Cas systems facilitates precise genetic modifications, enabling phages to serve as carriers of functional genes/proteins, thereby enhancing diagnostics, drug delivery, and therapy. Phage engineering holds promise in transforming precision medicine, addressing antibiotic resistance, and advancing diverse applications. Emphasizing the profound therapeutic potential of phages, this review underscores their pivotal role in combatting bacterial diseases and highlights their significance in the post-antibiotic era.

Genetic Engineering and Biosynthesis Technology: Keys to Unlocking the Chains of Phage Therapy.

Phages possess the ability to selectively eliminate pathogenic bacteria by recognizing bacterial surface receptors. Since their discovery, phages have been recognized for their potent bactericidal properties, making them a promising alternative to antibiotics in the context of rising antibiotic resistance. However, the rapid emergence of phage-resistant strains (generally involving temperature phage) and the limited host range of most phage strains have hindered their antibacterial efficacy, impeding their full potential. In recent years, advancements in genetic engineering and biosynthesis technology have facilitated the precise engineering of phages, thereby unleashing their potential as a novel source of antibacterial agents. In this review, we present a comprehensive overview of the diverse strategies employed for phage genetic engineering, as well as discuss their benefits and drawbacks in terms of bactericidal effect.

Cell Free Bacteriophage Synthesis from Engineered Strains Improves Yield.

Phage therapy to treat life-threatening drug-resistant infections has been hampered by technical challenges in phage production. Cell-free bacteriophage synthesis (CFBS) can overcome the limitations of standard phage production methods by manufacturing phage virions in vitro. CFBS mimics intracellular phage assembly using transcription/translation machinery (TXTL) harvested from bacterial lysates and combined with reagents to synthesize proteins encoded by a phage genomic DNA template. These systems may enable rapid phage production and engineering to accelerate phages from bench-to-bedside. TXTL harvested from wild type or commonly used bacterial strains was not optimized for bacteriophage production. Here, we demonstrate that TXTL from genetically modified E. coli BL21 can be used to enhance phage T7 yields in vitro by CFBS. Expression of 18 E. coli BL21 genes was manipulated by inducible CRISPR interference (CRISPRi) mediated by nuclease deficient Cas12a from F. novicida (dFnCas12a) to identify genes implicated in T7 propagation as positive or negative effectors. Genes shown to have a significant effect were overexpressed (positive effectors) or repressed (negative effectors) to modify the genetic background of TXTL harvested for CFBS. Phage T7 CFBS yields were improved by up to 10-fold in vitro through overexpression of translation initiation factor IF-3 (infC) and small RNAs OxyS and CyaR and by repression of RecC subunit exonuclease RecBCD. Continued improvement of CFBS will mitigate phage manufacturing bottlenecks and lower hurdles to widespread adoption of phage therapy.

Engineered lytic phage of Bacillus cereus and its application in milk

Phages have been approved for use in the food industry to control bacterial contamination in some countries. However, their broader adoption is hindered by some limitations. For instance, the persistence of infectious phages in the food industry can lead to the emergence of resistant bacteria, which negatively impacts the long-term effectiveness of phages. Additionally, the narrow host range of phages limits their effectiveness against various strains. To address these deficiencies, phage engineering has been proposed as a rational approach for modifying phages. In this study, we developed a simple and efficient engineering method for Bacillus cereus phage, using DK1 as an example, to reduce the number of residual phages and expand its range of hosts. Specifically, we knocked out the appendage gene, which codes for the receptor-binding protein, to produce phage progeny with structural defects in their appendages, resulting in the loss of infectivity after host elimination. Furthermore, we used plasmid-mediated means to express different appendage proteins during phage preparation, which allowed altering the host spectrum of the engineered phages without gene insertion. In practical applications, our engineered phages effectively reduced the number of B. cereus in milk and prevented the amplification of active progeny. Our strategy transformed phages from active viruses into more controllable antibacterial agents, making them safer and more efficient for the prevention and control of B. cereus. Moreover, we believe this strategy will help drive the use of engineered phages in the food industry.

Bacteriophage genome engineering for phage therapy to combat bacterial antimicrobial resistance as an alternative to antibiotics.

Bacteriophages (phages) are viruses that mainly infect bacteria and are ubiquitously distributed in nature, especially to their host. Phage engineering involves nucleic acids manipulation of phage genome for antimicrobial activity directed against pathogens through the applications of molecular biology techniques such as synthetic biology methods, homologous recombination, CRISPY-BRED and CRISPY-BRIP recombineering, rebooting phage-based engineering, and targeted nucleases including CRISPR/Cas9, zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Management of bacteria is widely achieved using antibiotics whose mechanism of action has been shown to target both the genetic dogma and the metabolism of pathogens. However, the overuse of antibiotics has caused the emergence of multidrug-resistant (MDR) bacteria which account for nearly 5million deaths as of 2019 thereby posing threats to the public health sector, particularly by 2050. Lytic phages have drawn attention as a strong alternative to antibiotics owing to the promising efficacy and safety of phage therapy in various models in vivo and human studies. Therefore, harnessing phage genome engineering methods, particularly CRISPR/Cas9 to overcome the limitations such as phage narrow host range, phage resistance or any potential eukaryotic immune response for phage-based enzymes/proteins therapy may designate phage therapy as a strong alternative to antibiotics for combatting bacterial antimicrobial resistance (AMR). Here, the current trends and progress in phage genome engineering techniques and phage therapy are reviewed.

Engineering bacteriophages for enhanced host range and efficacy: insights from bacteriophage-bacteria interactions.

Bacteriophages, the most abundant organisms on earth, have the potential to address the rise of multidrug-resistant bacteria resulting from the overuse of antibiotics. However, their high specificity and limited host range can hinder their effectiveness. Phage engineering, through the use of gene editing techniques, offers a means to enhance the host range of bacteria, improve phage efficacy, and facilitate efficient cell-free production of phage drugs. To engineer phages effectively, it is necessary to understand the interaction between phages and host bacteria. Understanding the interaction between the receptor recognition protein of bacteriophages and host receptors can serve as a valuable guide for modifying or replacing these proteins, thereby altering the receptor range of the bacteriophage. Research and development focused on the CRISPR-Cas bacterial immune system against bacteriophage nucleic acids can provide the necessary tools to promote recombination and counter-selection in engineered bacteriophage programs. Additionally, studying the transcription and assembly functions of bacteriophages in host bacteria can facilitate the engineered assembly of bacteriophage genomes in non-host environments. This review highlights a comprehensive summary of phage engineering methods, including in-host and out-of-host engineering, and the use of high-throughput methods to understand their role. The main aim of these techniques is to harness the intricate interactions between bacteriophages and hosts to inform and guide the engineering of bacteriophages, particularly in the context of studying and manipulating the host range of bacteriophages. By employing advanced high-throughput methods to identify specific bacteriophage receptor recognition genes, and subsequently introducing modifications or performing gene swapping through in-host recombination or out-of-host synthesis, it becomes possible to strategically alter the host range of bacteriophages. This capability holds immense significance for leveraging bacteriophages as a promising therapeutic approach against antibiotic-resistant bacteria.

Engineered Phage-Based Cancer Vaccines: Current Advances and Future Directions.

Bacteriophages have emerged as versatile tools in the field of bioengineering, with enormous potential in tissue engineering, vaccine development, and immunotherapy. The genetic makeup of phages can be harnessed for the development of novel DNA vaccines and antigen display systems, as they can provide a highly organized and repetitive presentation of antigens to immune cells. Bacteriophages have opened new possibilities for the targeting of specific molecular determinants of cancer cells. Phages can be used as anticancer agents and carriers of imaging molecules and therapeutics. In this review, we explored the role of bacteriophages and bacteriophage engineering in targeted cancer therapy. The question of how the engineered bacteriophages can interact with the biological and immunological systems is emphasized to comprehend the underlying mechanism of phage use in cancer immunotherapy. The effectiveness of phage display technology in identifying high-affinity ligands for substrates, such as cancer cells and tumor-associated molecules, and the emerging field of phage engineering and its potential in the development of effective cancer treatments are discussed. We also highlight phage usage in clinical trials as well as the related patents. This review provides a new insight into engineered phage-based cancer vaccines.