scholarly journals Engineering Phage Host-Range and Suppressing Bacterial Resistance through Phage Tail Fiber Mutagenesis

Cell ◽  
2019 ◽  
Vol 179 (2) ◽  
pp. 459-469.e9 ◽  
Author(s):  
Kevin Yehl ◽  
Sébastien Lemire ◽  
Andrew C. Yang ◽  
Hiroki Ando ◽  
Mark Mimee ◽  
...  
Keyword(s):  
Host Range ◽  
Bacterial Resistance ◽  
Tail Fiber ◽  
Phage Tail

scholarly journals Engineering Phage Host-Range and Suppressing Bacterial Resistance Through Phage Tail Fiber Mutagenesis

2019 ◽  
Author(s):  
Kevin Yehl ◽  
Sébastien Lemire ◽  
Andrew C. Yang ◽  
Hiroki Ando ◽  
Mark Mimee ◽  
...  
Keyword(s):  
Host Range ◽  
Bacterial Resistance ◽  
Mouse Skin ◽  
Infection Model ◽  
Tail Fiber ◽  
List Type ◽  
Phage Tail ◽  
Genetically Engineer

SUMMARYThe rapid emergence of antibiotic-resistant infections is prompting increased interest in phage-based antimicrobials. However, acquisition of resistance by bacteria is a major issue in the successful development of phage therapies. Through natural evolution and structural modeling, we identified host-range determining regions (HRDR) in the T3 phage tail fiber protein and developed a high-throughput strategy to genetically engineer these regions through site-directed mutagenesis. Inspired by antibody specificity engineering, this approach generates deep functional diversity (>107 different members), while minimizing disruptions to the overall protein structure, resulting in synthetic “phagebodies”. We showed that mutating HRDRs yields phagebodies with altered host-ranges. Select phagebodies enable long-term suppression of bacterial growth by preventing the appearance of resistance in vitro and are functional in vivo using a mouse skin infection model. We anticipate this approach may facilitate the creation of next-generation antimicrobials that slow resistance development and could be extended to other viral scaffolds for a broad range of applications.HighlightsVastly diverse phagebody libraries containing 107 different members were created.Structure-informed engineering of viral tail fibers efficiently generated host-range alterations.Phagebodies prevented the development of bacterial resistance across long timescales in vitro and are functional in vivo.

scholarly journals Evaluating Phage Tail Fiber Receptor-Binding Proteins Using a Luminescent Flow-Through 96-Well Plate Assay

2021 ◽  
Vol 12 ◽  
Author(s):  
Emma L. Farquharson ◽  
Ashlyn Lightbown ◽  
Elsi Pulkkinen ◽  
Téa Russell ◽  
Brenda Werner ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Host Range ◽  
Infection Process ◽  
Bacterial Disease ◽  
Tail Fiber ◽  
Long Tail ◽  
Bacterial Surface ◽  
Specific Phage ◽  
Phage Tail ◽  
Tail Fibers

Phages have demonstrated significant potential as therapeutics in bacterial disease control and as diagnostics due to their targeted bacterial host range. Host range has typically been defined by plaque assays; an important technique for therapeutic development that relies on the ability of a phage to form a plaque upon a lawn of monoculture bacteria. Plaque assays cannot be used to evaluate a phage’s ability to recognize and adsorb to a bacterial strain of interest if the infection process is thwarted post-adsorption or is temporally delayed, and it cannot highlight which phages have the strongest adsorption characteristics. Other techniques, such as classic adsorption assays, are required to define a phage’s “adsorptive host range.” The issue shared amongst all adsorption assays, however, is that they rely on the use of a complete bacteriophage and thus inherently describe when all adsorption-specific machinery is working together to facilitate bacterial surface adsorption. These techniques cannot be used to examine individual interactions between a singular set of a phage’s adsorptive machinery (like long tail fibers, short tail fibers, tail spikes, etc.) and that protein’s targeted bacterial surface receptor. To address this gap in knowledge we have developed a high-throughput, filtration-based, bacterial binding assay that can evaluate the adsorptive capability of an individual set of a phage’s adsorption machinery. In this manuscript, we used a fusion protein comprised of an N-terminal bioluminescent tag translationally fused to T4’s long tail fiber binding tip (gp37) to evaluate and quantify gp37’s relative adsorptive strength against the Escherichia coli reference collection (ECOR) panel of 72 Escherichia coli isolates. Gp37 could adsorb to 61 of the 72 ECOR strains (85%) but coliphage T4 only formed plaques on 8 of the 72 strains (11%). Overlaying these two datasets, we were able to identify ECOR strains incompatible with T4 due to failed adsorption, and strains T4 can adsorb to but is thwarted in replication at a step post-adsorption. While this manuscript only demonstrates our assay’s ability to characterize adsorptive capabilities of phage tail fibers, our assay could feasibly be modified to evaluate other adsorption-specific phage proteins.

scholarly journals DNA Inversion in the Tail Fiber Gene Alters the Host Range Specificity of Carotovoricin Er, a Phage-Tail-Like Bacteriocin of Phytopathogenic Erwinia carotovora subsp.carotovora Er

2001 ◽  
Vol 183 (21) ◽  
pp. 6274-6281 ◽  
Author(s):  
Hoa Anh Nguyen ◽  
Toshio Tomita ◽  
Morihiko Hirota ◽  
Jun Kaneko ◽  
Tetsuya Hayashi ◽  
...  
Keyword(s):  
Host Range ◽  
Erwinia Carotovora ◽  
Tail Fiber ◽  
Chromosomal Dna ◽  
Tail Fiber Protein ◽  
Dna Inversion ◽  
Phage Tail ◽  
Fiber Protein ◽  
Invertase Gene ◽  
Fiber Gene

ABSTRACT Carotovoricin Er is a phage-tail-like bacteriocin produced byErwinia carotovora subsp. carotovorastrain Er, a causative agent for soft rot disease in plants. Here we studied binding and killing spectra of carotovoricin Er preparations for various strains of the bacterium (strains 645Ar, EC-2, N786, and P7) and found that the preparations contain two types of carotovoricin Er with different host specificities; carotovoricin Era possessing a tail fiber protein of 68 kDa killed strains 645Ar and EC-2, while carotovoricin Erb with a tail fiber protein of 76 kDa killed strains N786 and P7. The tail fiber proteins of 68 and 76 kDa had identical N-terminal amino acid sequences for at least 11 residues. A search of the carotovoricin Er region in the chromosome of strain Er indicated the occurrence of a DNA inversion system for the tail fiber protein consisting of (i) two 26-bp inverted repeats inside and downstream of the tail fiber gene that flank a 790-bp fragment and (ii) a putative DNA invertase gene with a 90-bp recombinational enhancer sequence. In fact, when a 1,400-bp region containing the 790-bp fragment was amplified by a PCR using the chromosomal DNA of strain Er as the template, both the forward and the reverse nucleotide sequences of the 790-bp fragment were detected. DNA inversion of the 790-bp fragment also occurred in Escherichia coli DH5α when two compatible plasmids carrying either the 790-bp fragment or the invertase gene were cotransformed into the bacterium. Furthermore, hybrid carotovoricin CGE possessing the tail fiber protein of 68 or 76 kDa exhibited a host range specificity corresponding to that of carotovoricin Era or Erb, respectively. Thus, a DNA inversion altered the C-terminal part of the tail fiber protein of carotovoricin Er, altering the host range specificity of the bacteriocin.

scholarly journals How to Train Your Phage: The Recent Efforts in Phage Training

Biologics ◽  
2021 ◽  
Vol 1 (2) ◽  
pp. 70-88
Author(s):  
Abdallah Abdelsattar ◽  
Alyaa Dawooud ◽  
Nouran Rezk ◽  
Salsabil Makky ◽  
Anan Safwat ◽  
...  
Keyword(s):  
Host Range ◽  
Pathogenic Bacteria ◽  
Bacterial Resistance ◽  
Phage Therapy ◽  
Current Knowledge ◽  
Lytic Replication ◽  
Resistant Bacteria ◽  
Infection Cycles ◽  
Different Strains ◽  
Specific Species

Control of pathogenic bacteria by deliberate application of predatory phages has potential as a powerful therapy against antibiotic-resistant bacteria. The key advantages of phage biocontrol over antibacterial chemotherapy are: (1) an ability to self-propagate inside host bacteria, (2) targeted predation of specific species or strains of bacteria, (3) adaptive molecular machinery to overcome resistance in target bacteria. However, realizing the potential of phage biocontrol is dependent on harnessing or adapting these responses, as many phage species switch between lytic infection cycles (resulting in lysis) and lysogenic infection cycles (resulting in genomic integration) that increase the likelihood of survival of the phage in response to external stress or host depletion. Similarly, host range will need to be optimized to make phage therapy medically viable whilst avoiding the potential for deleteriously disturbing the commensal microbiota. Phage training is a new approach to produce efficient phages by capitalizing on the evolved response of wild-type phages to bacterial resistance. Here we will review recent studies reporting successful trials of training different strains of phages to switch into lytic replication mode, overcome bacterial resistance, and increase their host range. This review will also highlight the current knowledge of phage training and future implications in phage applications and phage therapy and summarize the recent pipeline of the magistral preparation to produce a customized phage for clinical trials and medical applications.

Nonlytic Recombinant Phage Tail Fiber Protein for Specific Recognition of Pseudomonas aeruginosa

2018 ◽  
Vol 90 (24) ◽  
pp. 14462-14468 ◽  
Author(s):  
Yong He ◽  
Yanli Shi ◽  
Mengli Liu ◽  
Yingran Wang ◽  
Lin Wang ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Tail Fiber ◽  
Tail Fiber Protein ◽  
Recombinant Phage ◽  
Specific Recognition ◽  
Phage Tail ◽  
Fiber Protein

scholarly journals Host Range Expansion of Pseudomonas Virus LUZ7 Is Driven by a Conserved Tail Fiber Mutation

PHAGE ◽  
2020 ◽  
Vol 1 (2) ◽  
pp. 87-90 ◽  
Author(s):  
Maarten Boon ◽  
Dominique Holtappels ◽  
Cédric Lood ◽  
Vera van Noort ◽  
Rob Lavigne
Keyword(s):  
Host Range ◽  
Range Expansion ◽  
Tail Fiber ◽  
Host Range Expansion

scholarly journals Efficiency of Phage φ6 for Biocontrol of Pseudomonas syringae pv. syringae: An in Vitro Preliminary Study

2019 ◽  
Vol 7 (9) ◽  
pp. 286 ◽  
Author(s):  
Larindja A. M. Pinheiro ◽  
Carla Pereira ◽  
Carolina Frazão ◽  
Victor M. Balcão ◽  
Adelaide Almeida
Keyword(s):  
Solar Radiation ◽  
Host Range ◽  
Pseudomonas Syringae ◽  
Bacterial Resistance ◽  
Phage Therapy ◽  
Bacterial Species ◽  
Fruit Trees ◽  
Range Analysis ◽  
Maximum Reduction

Pseudomonas syringae is a plant-associated bacterial species that has been divided into more than 60 pathovars, with the Pseudomonas syringae pv. syringae being the main causative agent of diseases in a wide variety of fruit trees. The most common treatments for biocontrol of P. syringae pv. syringae infections has involved copper derivatives and/or antibiotics. However, these treatments should be avoided due to their high toxicity to the environment and promotion of bacterial resistance. Therefore, it is essential to search for new approaches for controlling P. syringae pv. syringae. Phage therapy can be a useful alternative tool to the conventional treatments to control P. syringae pv. syringae infections in plants. In the present study, the efficacy of bacteriophage (or phage) φ6 (a commercially available phage) was evaluated in the control of P. syringae pv. syringae. As the plants are exposed to the natural variability of physical and chemical parameters, the influence of pH, temperature, solar radiation and UV-B irradiation on phage φ6 viability was also evaluated in order to develop an effective phage therapy protocol. The host range analysis revealed that the phage, besides its host (P. syringae pv. syringae), also infects the Pseudomonas syringae pv. actinidiae CRA-FRU 12.54 and P. syringae pv. actinidiae CRA-FRU 14.10 strains, not infecting strains from the other tested species. Both multiplicities of infection (MOIs) tested, 1 and 100, were effective to inactivate the bacterium, but the MOI 1 (maximum reduction of 3.9 log CFU/mL) was more effective than MOI 100 (maximum reduction of 2.6 log CFU/mL). The viability of phage φ6 was mostly affected by exposure to UV-B irradiation (decrease of 7.3 log PFU/mL after 8 h), exposure to solar radiation (maximum reduction of 2.1 PFU/mL after 6 h), and high temperatures (decrease of 8.5 PFU/mL after 6 days at 37 °C, but a decrease of only 2.0 log PFU/mL after 67 days at 15 °C and 25 °C). The host range, high bacterial control and low rates of development of phage-resistant bacterial clones (1.20 × 10−3) suggest that this phage can be used to control P. syringae pv. syringae infections in plants, but also to control infections by P. syringae pv. actinidiae, the causal agent of bacterial canker of kiwifruit. Although the stability of phage φ6 was affected by UV-B and solar radiation, this can be overcome by the application of phage suspensions at the end of the day or at night.

scholarly journals Paving the Way to Unveil the Diversity and Evolution of Phage Genomes

Viruses ◽  
2020 ◽  
Vol 12 (9) ◽  
pp. 905
Author(s):  
Alejandro Reyes ◽  
Martha J. Vives
Keyword(s):  
Molecular Evolution ◽  
Host Range ◽  
Molecular Mechanisms ◽  
Bacterial Resistance ◽  
Viral Pathogenesis ◽  
Resistance Mechanisms ◽  
Molecular Tools ◽  
Special Issue ◽  
Bacterial Host ◽  
Host Interactions

Phage biology has been developing for the last hundred years, and the potential of phages as tools and treatments has been known since their early discovery. However, the lack of knowledge of the molecular mechanisms coded in phage genomes hindered the development of the field. With current molecular methods, the last decade has been a resurgence of the field. The Special Issue on “Diversity and Evolution of Phage Genomes” is a great example with its 17 manuscripts published. It covers some of the latest methods to sample and characterize environmental and host associated viromes, considering experimental biases and computational developments. Furthermore, the use of molecular tools coupled with traditional methods has allowed to isolate and characterize viruses from different hosts and environments with such diversity that even a new viral class is being proposed. The viruses described cover all different phage families and lifestyles. However, is not only about diversity; the molecular evolution is studied in a set of manuscripts looking at phage-host interactions and their capacity to uncover the frequency and type of mutations behind the bacterial resistance mechanisms and viral pathogenesis, and such methods are opening new ways into identifying potential receptors and characterizing the bacterial host range.

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