Can lysogenic phages be trained to enter into the lytic cycle?

Have you ever come across the word phage training?

Phages are viruses that eat up bacteria; that's what many of us know. They are the most abundant biological entities in the environment. They live close to the bacteria they eat up and can evolve with evolving bacteria. Once phages enter into bacteria, they either undergo the lytic cycle, which involves the lysis of the bacteria, or the lysogenic cycle, which consists of integrating the phage genome into that of the bacterial. The lytic phages are highly utilized in the therapeutic field and biocontrol systems to curb many bacteria. However, the emergency of resistant bacteria against antibiotics and phages themselves has necessitated the need for scientists to train phages.

Optimizing bacteriophage engineering. Photo by Favor et al. (2020)

Phage training is an emerging protocol in the production of ef´Čücient phages that can act against a broad host range of bacteria, especially those resistant to phages. Training phages can be done by utilizing their natural capacity to evolve to counter resistance developed by bacteria, a significant barrier to phage therapy. Training phages is usually by coevolving them with their host, which enhances their capacity for suppressing bacterial growth and causes a delay in the emergence of resistance. Suppression of bacterial growth is mainly brought about by several mechanisms, which is vital in producing a robust therapeutic in bacterial treatment. The ability to overcome bacterial resistance mechanisms can be through phage adaptation.

Phage training and the Appelmans protocol (phage training protocol)

Phage adaptation to bacterial hosts, also known as phage training, can be best understood through co-evolution studies that employ the Appelmans protocol. In this protocol, the training of phages is divided into natural, enforced, and engineered procedures. 

Natural phage training

There is no bias between the selection of bacteria and phage; both are developed on the same track to keep the balance that exists in nature without human interference. 

Engineered phage training

The selection is absolutely biased in engineered phage training as it evolves naturally after engineering. 

Enforced phage training

However, in enforced phage training, the selection is biased toward the phage, for example, by countering phage-resistant secondary bacterial growth through co-treatment with silver nanoparticles or antibiotics. Implemented phage training can be through pseudo-enforced phage training approaches where the effect is temporary and dependent upon the effector. For example, using antibiotics to convert the lysogenic cycle to lytic in temperate phage and enforced phage training where the induced changes are permanent, exposing the phage to a chelating agent. 
Successful trials of training different strains of phages to switch into lytic replication mode, overcome bacterial resistance, and increase their host range have been reported. The current knowledge of phage training will have implications in phage applications and phage therapy against a wide range of resistant bacteria.

Training lysogenic phages to be lytic 

Training phages to switch from the lysogenic cycle to the lytic cycle involves controlling the replication dynamics of prophages and thence enabling their application to eliminate pathogenic bacteria. 
Training phages from lysogenic cycle to lytic cycle involves the application of lysis-inducing agents such as Mitomycin-C, then incubating bacteria for several hours around 7 h, and finally studying the outcome. Phage particles are then detected using an electron microscope, and an extrachromosomal band, indicative of phage nucleic acid, is visualized by gel electrophoresis. This procedure was employed in studying the phages of Serpulina hyodysenteriae.
Temperate phages can also be induced to switch to the lytic cycle through incubation with sodium pyrophosphate, which causes mutations resulting in viable lytic phages. This procedure was done to SA13 phages that target Staphylococcus aureus bacteria where random mutations were observed that resulted in non-functional truncated genes in SA13m, including integrase, CI repressor, and anti-repressor protein. The virulent mutant SA13m did not show lysogens and could lyse bacterial cells faster than the wild-type.
Prophage can also be induced to shift to the lytic cycle by culturing in sub-optimal acidic culture media (pH 5), and then the increase in the number of non-viable bacterial cells and the number of virus-like particles (VLPs) when observed indicates that the phage has shifted into a lytic mode of action. Increasing chlorine (IV) concentration from 0.002 to 0.1 mM also increases the virus-like particles hence switching prophage to the lytic cycle. This makes it possible to use prophage under acidic conditions and even in the presence of a low level of chlorine to control pathogenic bacteria.

Training lytic phages to be lysogenic 

The training of phages to become lysogenic can be done by inducing a phage to switch its replication mode from the lytic to the lysogenic cycle and inhibiting lysis during the superinfection exclusion (Sie). The Sie occurs when phages are not allowed to enter the host cell due to the presence of the same or closely related phages within the bacteria. The lytic to lysogenic switch is favored by a high multiplicity of infection (MOI) and infection by more than one replicating phage.
Phage training to become lysogenic can be done by increasing the initial phage titer, which elevates the expression of transcription factors and consequently increases CII gene expression (CII is a working gene in the lysogenic decision gene cluster). CII gene upregulates the CI gene, the primary agent in the phage lysogenization process. Phage training to become lysogenic can also be done by adjusting the initial inoculum by controlling the phage behaviors between lytic and lysogenic cycles. A larger inoculum of phages or lower inoculum of bacteria is employed.

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