Team:Grenoble-Alpes/phage lysis
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DNA EXTRACTION
The first main step in our system is the lysis of bacteria by bacteriophages. This step is decisive for all the rest of the system. In fact this step allows the release of DNA in the medium by lysing the specific bacteria. It is this specific DNA that will be extracted, digested and detected by our plasmid probes. So if the DNA released hybridize to the probes, it gives two essential information:
- Which bacterial species are present in the patient sample.
- What are the best bacteriophages capable of infecting this bacteria, and thus, treating the patient.
About bacteriophages
But to understand a little more this last point, let’s mention what is a bacteriophage and how it infects bacteria: Bacteriophages, also called phages, are bacteria-specific viruses: each bacteriophage is able to infect one or more bacterial strains from a single bacterial species. This specificity is characterized by differences in infection and replication efficiency. This last feature is proportional to the number of lysed bacteria and therefore to the amount of DNA released which will be able to hybridize with the probes. That is how, after the transformation step, we will be able to determine the lysis efficiency by following the fluorescence rate measured by our system. Then we will be able to choose the best bacterium killer bacteriophage.
However, be careful: some bacteriophages are not just able to lyse bacteria. Indeed, as you see in Figure 1, two types of infection cycles are distinguished:
1) Lytic cycle: the bacteriophage, once inside the bacterial host, uses the machinery of the infected cell to replicate, releasing a great number of new virions after a bacterial burst (in red in Figure 1).
2) Lysogenic cycle: the bacteriophage, once inside the bacterial host, integrates its DNA to the host DNA and becomes a prophage able to transmit itself to the descendants (in blue in Figure 1).
Lytic cycle
Lytic bacteriophages, as their name suggests, destroy the bacteria. They divert the bacterial machinery to their advantage to reproduce and multiply.
Indeed, bacteriophages, unable to reproduce by their own means, inject their genetic material into host bacteria (red 1. in Figure 1). Thanks to the enzymes and ribosomes of the host, the viral genome can be replicated and translated to form many copies (red 3. in Fig. 1). At the end of the process, the bacteria burst and dozens or even hundreds new bacteriophages - identical to the original - are released in the medium and therefore available to attack other bacteria of the same species (red 4. in the Fig. 1).
True “professional killers”, lytic bacteriophages are the natural predators of bacteria. It is precisely these lytic bacteriophages that are used for therapeutic purposes to fight against bacterial infections (bacteriophage therapy). For example, in Russia, bacteriophages are used to treat foot infections in patients with type 2 diabetes, known as diabetic foot ulcers and may cause amputation ; as described in the scientific article Bacteriophage Treatment of Infected Diabetic Foot Ulcers by scientists of the Institute of Chemical Biology and Fundamental Medicine SB RAS, Laboratory of Molecular Microbiology, Novosibirsk, Russian Federation. Thereby, we also use lytic bacteriophages in our system.
Lysogenic cycle
Temperate bacteriophages have the property of integrating their genome within the bacterial chromosomes. This phenomenon is called transduction and the bacteriophage cycle is called the lysogenic cycle. Bacteriophages can thus confer new properties to the bacterium, beneficial or not (e.g. virulences genes). For example, lysogenic bacteriophages can “capture” in their genome bacterial genes of interest (resistance to antibiotics, virulence genes…) that they can subsequently propagate within species, or from a species to the other. As a result, bacteriophages contribute to the diversification of bacterial species.
They can remain quiescent for a long time - such as “dormant agents”, carpeted in the bacterial genome (then called prophages) before starting their reproduction and entering a lytic cycle. These temperate bacteriophages are not used in therapy. During the production of bacteriophages for a therapeutic use, one must even ensure the absence of temperate bacteriophage in the preparation. Although bacteriophages cannot recognize human cells and thus they are considered harmless for human beings, it’s necessary to be careful because bacteriophages are potential gene vectors. These genes can be transferred horizontally to other organism and then, be dangerous to the patient.
In contrast, temperate bacteriophages are valuable adjuncts of molecular biology laboratories, which use them as tools to implant genes into bacteria to modify the genome and allow (for example) the manufacture of molecules by the bacterium, used as “factory”.
How to get a very high titer of bacteriophages ?
So now, we know how infection cycle works and the importance of bacteriophages. But before testing them with their bacterial hosts and extracting DNA, bacteriophages must be prepared! To do this we have to realize 3 steps:
- bacteriophage amplification [see protocol here]
- bacteriophage purification/concentration [see protocol here]
- plaque assay to determine the bacteriophage titer [see protocol here]
The bacteriophage amplification allows us to get a very high titer of bacteriophages. So we will be able to know if the phage titer influences the lysis efficiency and the amount of extracted DNA. To amplify bacteriophages it is enough to put them with their bacterial host in a liquid medium. To facilitate bacteriophage adsorption we add MgCl2 and CaCl2. Then, bacteriophages infect the bacterial culture and replicate themselves in an impressive number until the death of all the bacteria (Fig. 2).
Once this done, it’s necessary to purify bacteriophages by removing everything that is not needed (bacterial debris in particular). For that, we use a NaCl solution which helps bacteriophages to detach from the cell wall and we centrifuge to pellet debris and to keep only the supernatant containing bacteriophages. Then we use a PEG/NaCl solution which helps bacteriophages to crystallize and, after another centrifugation, to pellet [see protocol here]. We can afterward resuspend them in an adequate buffer. This step enables to concentrate the amplified phages and to store them (Fig. 3). The used buffer is named SM Buffer according to the Sodium chloride and the Magnesium sulfate that compose it [see protocol here]. These elements allow the bacteriophages to stabilize. Then they could be stored at 4°C for a long time.