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Phage Engineering: History
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Subjects: Biotechnology & Applied Microbiology
Contributor: Zhi Liu

Along with the excessive use of antibiotics, the emergence and spread of multidrug-resistant bacteria has become a public health problem and a great challenge vis-à-vis the control and treatment of bacterial infections. As the natural predators of bacteria, phages have reattracted researchers’ attentions. Phage therapy is regarded as one of the most promising alternative strategies to fight pathogens in the post-antibiotic era. Recently, genetic and chemical engineering methods have been applied in phage modification. Among them, genetic engineering includes the expression of toxin proteins, modification of host recognition receptors, and interference of bacterial phage-resistant pathways. Chemical engineering, meanwhile, involves crosslinking phage coats with antibiotics, antimicrobial peptides, heavy metal ions, and photothermic matters. Those advances greatly expand the host range of phages and increase their bactericidal efficiency, which sheds light on the application of phage therapy in the control of multidrug-resistant pathogens.

  • phage engineering
  • multidrug resistance
  • virulence gene
  • CRISPR–Cas system
  • tail fiber mutant
  • nanoparticles
  • phage immobilization

1. introduction

In recent years, multidrug-resistant bacterial infections have emerged as one of the most challenging global public health threats, causing severe influences on food safety, environmental ecology, and the social economy[1][2]. Multidrug resistance (MDR) genes derived from gene mutation and gene transfer have also greatly challenged the usage of last-line antibiotic therapy, including carbapenem and polymyxin [3][4]. At present, more than 400 different types and 20,000 potential MDR genes have been predicted from the sequenced bacterial genomic data, which particularly deserves alertness[5]. More seriously, the development of novel antibiotics has become increasingly difficult due to antibiotic resistance. Meanwhile, many pharmaceutical companies have not invested in new antibiotics research and development due to a limited market profit[6]. Therefore, there is a pressing need to search for alternative strategies for MDR bacteria treatment. Phages, natural enemies of bacteria, are considered as a very promising therapy strategy [7]. The first phage research was conducted by Twort, who made an unusual observation about Staphylococcus lysis in 1915, which was 13 years before Fleming discovered penicillin, the first antibiotic [8]. Since then, investigators have successively reported many different special viruses in mycoplasmas, spirochetes, actinomycetes, and cyanobacteria [9]. At the onset of phage discovery, they were used to treat bacterial infections, and the initial results were promising [10]. However, the application of phage therapy was limited for its narrow host range and the range of antiviral strategies evolved in bacteria [10][11][12]. Interestingly, phage therapy gradually faded away in the 1940s due to the emergence of antibiotics, yet it has recently regained interest in the wake of the emergence of antibiotic-resistant bacteria. To better cure infections, researchers have developed different phage therapy strategies, including phage cocktails and the combination of phages with other drugs [13][14][15]. More importantly, many researchers have attempted to develop novel engineered phages through genetic and chemical approaches. These artificially armed phages could improve the antibacterial efficacy against MDR bacteria by interfering with their drug-resistant pathways (or in other ways) with little disturbance to the whole microenvironment [16][17].

2. Phage Genetic Modification

The genetic modification of phages mainly includes the gene mutation, gene replacement, and gene integration of foreign genes using molecular techniques in order to expand the host range or enhance the antibacterial effect of the phages. Gene mutation and replacement usually occur in the genes related to the tail fiber protein to broaden the phage host range. While the integration of foreign genes often integrates some genes into the phage genome nonfunctional region, the products that integrate genes are usually harmful to the host. We summarized the previous articles on the genetic modifications of phages and sorted the results in Table 1 according to the types of phages modified, the modified genes, and the antibacterial mechanisms.

Table 1. Genetic engineering methods applied in phage modification.

Phage

Genetic Modification

Mechanism

Goal/Target

Ref.

M13

λS105; Bgl Ⅱ

Membrane damage; DNA breakage

To reduce endotoxin

[18]

M13

Gef; ChpBK

Membrane damage; mRNA degradation

To increase bactericidal efficiency

[19]

T7Select

peptide 1018

Kill cells; inhibit biofilm

Biofilm

[12]

M13mp18

LexA3

Suppress SOS system

Antibiotic-resistant bacteria

[20]

Wild-type T7

DspB

Hydrolysis β-1,6-N-acetyl-D-glucosamine

Biofilm

[21]

T7Select415-1

AiiA

Inhibit quorum sensing

Biofilm

[22]

M13 phagemid

CRISPR-cas9

Target resistance genes

Antibiotic-resistant bacteria

[17]

M13 phagemid

CRISPR-cas9

Target resistance genes and virulent genes

Antibiotic-resistant bacteria

[16]

λ phage

CRISPR-cas3

Target resistance genes

Antibiotic-resistant bacteria

[23]

φ SaBov

CRISPR-cas9

Target the nuc gene

Antibiotic-resistant bacteria

[24]

T2, T3, Fd

Tail fiber genes

Expand the host range

Antiphage bacteria

[25][26][27][28]

3. Phage Chemical Modification

The fast growth of the current chemical industry has created numerous novel substances that did not exist before. These substances are entirely new for microorganisms and may function on bacteria with varied mechanisms. In this case, microorganisms have not evolved to generate enough resistance genes to respond to the impact of these new materials [29]. In this section, we will focus on phage chemical modification and summarize our findings in Table 2, wherein several novel chemical materials for modifying phages are presented, including silver nanoparticles (AgNPs), AIEgens, pheophorbide a (PPA), cellulose membrane, and indium tin oxide (ITO).

Table 2. Chemical engineering methods applied for phage modification. EDC/NHS: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydro/N-hydroxysuccinimide.

Phage

Chemical Modification

Binding Force

Ref

M13

Silver nanoparticles (AgNPs)

Ionic binding

[30]

PAP

AIEgens

Amide bond

[31]

JM phage

Pheophorbide a (PPA)

EDC/NHS Crosslinking

[32]

Bacteriophage T4 (ATCC 11303-B4)

Indium tin oxide (ITO)

Ionic binding

[33]

Phage cocktail

Cellulose membrane

Ionic binding

[34]

4. Challenges in Phage Therapy

As the antibiotic resistance crisis becomes more serious, alternative treatments for bacterial infections are urgently needed. Phages are one of the most promising antibacterial agents for clinical use [35]. However, phage therapy is limited by its unstable therapeutic efficacy caused by its narrow host range and bacterial resistance against phages. Interestingly, mounting evidence has demonstrated that engineered phages might be effective for treating bacterial infections. However, the lack of research regarding its effectiveness and safety has naturally limited the clinical or large-scale applications for genetically modified phages. Therefore, the effectiveness and safety tests of engineered phages are urgently needed in further clinical trials.

First of all, phages are strictly host-specific. Accordingly, phage therapy can target certain pathogens without disturbing the gut microbial ecology. However, when the host is infected by various pathogens, single-phage therapy cannot work effectively. Moreover, the sensitive phage needs to be identified according to the isolates that cause the infections, which is a time-consuming process and not suitable for acute infections. A variety of strategies can be used to solve this problem, including the use of a phage cocktail, a modified phage, and the combination of phages and antibiotics.

Secondly, the resistance of bacteria to a phage also results in poor treatment effects of phage therapy. Although some engineering strategies are designed to overcome bacteria resistances to phages and have yielded promising results, bacteria could also evolve themselves to resist those engineered phages gradually, which will thus decrease the efficacy of the engineered phage therapy.

Phages need to reach the infected lesions and adhere to the surface of pathogenic bacteria to exert their functions, so the effective concentrations in the lesions affect their efficacy. In clinical trials, phages cannot reach the lesions or maintain effective concentrations in the lesions due to their limited stability. Besides, most phages could be neutralized by human antibodies [36][37] and cleared by the reticuloendothelial system [74]. In order to ensure that phages can reach the lesions and maintain an effective concentration, it is necessary to determine the time, route, dose, and frequency of administration according to the type of phage and infected bacteria, as well as the different parts of the lesion, so as to determine the best treatment regimen [38].

Phages are distinguished from antibiotics and other chemical antimicrobial agents by their self-replicating features, which makes the pharmacokinetics of phages complicated [39]. Therefore, the determination of phage therapy for different pathogens will only become more complex and difficult.

Some improvements have been made for engineered phages to overcome these obstacles[40][41][42], so that engineered phages can reach the lesion sites and maintain effective concentrations in a certain period of time. However, the lack of clinical data is still a serious problem for the widespread application of engineered phages. Therefore, there is an urgent need for future clinical trials to be conducted that evaluate the effectiveness of engineered phages.

The safety of phage therapy cannot be ignored during clinical treatment. Phages can induce immune responses during phage therapy, causing inflammation [43]. The bacterial cell wall fragments, endotoxins, and enterotoxins, which contain impure phage preparations released from the lysis of pathogenic bacteria, may also stimulate the immune system and trigger local acute inflammatory responses[35][44][45][46]. Phages may cause damage to the body, because they carry toxin genes or induce gene mutations [43].

It is also necessary to set standards to evaluate the safety of phage therapy. The Food and Drug Administration (FDA) has set standards based on small-scale experiments that have evaluated the efficacy and safety of phage therapy [47]. Moreover, some standards have been based on the Quality by Design (QbD) and European Union Tissue and Cell Directives (EUTCD), which were established by 32 bacteriophage experts from 12 countries to evaluate the quality and safety of phage therapy products [48]. However, these evaluation systems mainly focus on natural phages. For the development of clinical trials, there is a need to develop a special evaluation standard for the safety evaluation of engineered phages.

 In addition to safety and effectiveness, scientists also face challenges when obtaining regulatory approval for phage therapy applications [49]. Currently, there is a lack of standardization and appropriate regulatory framework for phage therapy [50]. Further improvements of the relevant systems and regulations will promote the widespread application of bacteriophages.

This entry is adapted from the peer-reviewed paper 10.3390/antibiotics10020202

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