Artificial viruses
The first synthetic bacteriophages have been created
Vera Sysoeva, N+1
Scientists from Scientists have created bacteriophages with a shortened genome and described them. This is the first successful attempt to create synthetic bacterial viruses. The authors hope that their research will help in the creation of new antibacterial substances. The work was published in the journal Scientific Reports (Pires et al., Designing P.aeruginosa synthetic phages with reduced genomes).
The developing antibiotic resistance around the world is one of the most terrible threats to human health. It forces researchers to look for alternative therapeutic approaches. In particular, scientists are considering the use of bacteriophages – viruses that infect bacteria. The idea itself is not new: the use of bacteriophages as medicines began back in the 1920s, when antibiotics were not yet available. Since then, bacteriophages – both natural and modified by genetically engineered methods - have been used as therapy, but have not become a full–fledged substitute for antibiotics. The situation develops this way for many reasons, including due to the lack of knowledge of many phages.
The genomes of bacteriophages are small, however, there are areas in them (quite large, up to 80 percent of the genome), the functions of which are still unknown to scientists. There is some possibility that proteins encoded by unknown genes may be harmful to humans. In addition, "whole" genomes are inconvenient for further manipulation, for example, adding genes necessary for therapeutic purposes. The removal of genes with unknown functions allowed it would leave only known and safe genes and make room for new ones.
Similar experiments on minimizing genomes have already been conducted on living objects. The works carried out under the guidance of biologist Craig Weinter (J. Craig Venter) to create a bacterium with a minimal genome have gained fame. Then scientists identified part of the bacteria's genes Mycoplasma mycoides as "not vital" and removed them, obtaining, in fact, a new synthetic organism. You can read more about this direction in synthetic biology in the material "Living wage".
In the new work, Portuguese scientists sought to create a synthetic bacteriophage that would infect Pseudomonas aeruginosa (Pseudomonas aeruginosa). At the moment, the treatment of infections caused by R. aeruginosa is quite difficult due to the high resistance of this bacterium to many antibiotics. WHO calls the development of drugs against Pseudomonas aeruginosa one of the priorities.
Researchers from the University of Minho, led by Joana Azeredo, took as a basis a bacteriophage infecting R.aeruginosa, which they isolated from wastewater. To begin with, scientists tested the natural abilities of this phage (it was called RE3). Out of 28 samples received from patients R.aeruginosa bacteriophage was able to hit seven. The researchers obtained images of the bacteriophage using transmission electron microscopy and sequenced its genome. It turned out that the genome of the PE3 phage consists of 43.5 thousand nucleotide bases of double-stranded DNA. Based on the results of computer analysis, the researchers suggested that there are 55 protein-coding sequences in the genome. The authors concluded that the bacteriophage described by them belongs to the Autographiviridae family.
Further, scientists have suggested that two gene modules can be cut from the genome of the bacteriophage PE3, which probably encode proteins: from the first to the fifth (gp1-gp5) and from the sixth to the twelfth (gp6-gp12). The authors of the work created three variants of the synthetic genome: in two they removed one of the modules, and in the third they removed both. Using PCR, the researchers amplified the remaining genes and connected them in yeast cells into an artificial yeast chromosome. The resulting synthetic bacteriophage genome was isolated from yeast and transformed by them into a host bacterium P aeruginosa to test the ability of the hereditary information of the genome phage to run a program for the assembly of viral particles.
The scheme of the experiment is VM. Figures from the article by Pires et al.
The experiment was successful: visible phage plaques formed on the cups with bacteria – places where the virus damaged the bacteria.
To better understand how gene knockout affected the bacteriophage, the authors compared its viability with the wild type. Firstly, the phage plaques have decreased in size.
From left to right: phage plaques formed by the wild type of bacteriophage RE3 and bacteriophages with deletions gp1-gp5, gp6-gp12 and gp1-gp12.
Secondly, not all synthetic bacteriophages were able to infect the same strains of Pseudomonas aeruginosa as their predecessor. Only phages with deletion of the gp6-gp12 module had a significant effect on 7 out of 28 clinical samples. aeruginosa, the rest infected only 4 strains. In addition, phages with gp6-gp12 deletion showed growth rates similar to PE3, while phages with gp1-gp5 and gp1-gp12 deletions lagged by five and fifteen minutes, respectively.
The growth curve of bacteriophages.
At the same time, the antibacterial effectiveness of bacteriophages was not affected by the manipulations carried out. This was found out during in vitro experiments, where bacteriophages were added to bacterial cultures in the exponential growth phase. The ratio of viral particles to cells was 1:5. All phage variants showed the same efficacy, no significant differences were observed (p>0.01). Two hours after the addition of phages, the number of living cells decreased by five orders of magnitude in all samples compared to the control group (p<0.01). After that, however, the number of living cells began to grow again, and 24 hours after the start of the experiment almost did not differ from the control group (p<0.01). This demonstrates the rapid development of phage-insensitive mutant bacteria.
The number of live P. aeruginosa cells depending on the time after the addition of bacteriophages.
The researchers also tested the therapeutic efficacy of synthetic bacteriophages in vivo on a Large wax moth (G. mellonella). After infection with bacteria, the insects were injected with solutions with bacteriophages. After 24, 48 and 72 hours, the survival rate in the control group was 20, 13 and 10 percent, respectively. The group treated with bacteriophages showed the best results (p < 0.05): their survival rate was 50 percent after 24 hours, and 30 percent after two and three days. At the same time, synthetic bacteriophages and their natural predecessor coped with their task equally well.
Scientists hope that the methods of synthetic biology in the future will allow us to quickly develop and create specialized bacteriophages for many bacterial species.
Bacteriophages have already helped doctors before: for example, the combined use of phages and antibiotics saved the patient from an antibiotic-resistant infection. aeruginosa, and modified phages may have cured mycoplasma infection. In addition, Australian scientists have described how the presence of bacteriophages causes the bacterium to become sensitive to antibiotics again.
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