Bacteriophage Lytic Activity

Most phages elaborate at least two proteins, one of which is a murein hydrolase, or lysin, and the other is a membrane protein, which is given the designation holin.
The function of the holin is to create a lesion in the cytoplasmic membrane through which the murein hydrolase passes to gain access to the murein layer.


Lysis of bacterial cell wall is based on the action of two phage encoded enzymes: holins and lysins.

Lysins are phage-encoded murein hydrolases that act on the bacterial host cell wall at the terminal stage of the phage reproduction cycle to release progeny phage.

These enzymes are also known as phage lysozymes, endolysins, or muralytic/mureolytic enzymes. Their action is tightly regulated by holins, by membrane arrest, and by conversion from their inactive state. Their N-terminal catalytic domains are able to target almost every possible bond in the peptidoglycan network, and their corresponding C-terminal cell wall binding domains target the enzymes to their substrate.
Owing to their specificity and high activity, endolysins have been employed for various in vitro and in vivo aims, in food science, in microbial diagnostics, and for treatment of experimental infections.

Clearly, phage lysins represent great tools for use in molecular biology, biotechnology and in medicine, and we are just beginning to tap this potential.There are at least two distinct mechanisms by which phages destroy the cell wall.

Bacteriophages with large genomes use a holin-endolysin system, while bacteriophages with small genomes encode a single lysis protein. Some single protein lysis systems inhibit cell wall synthesis and are thus the phage analogs of antibiotics like penicillin. Sometimes also phage capsid proteins are responsible for lysis.

In gram-positive bacteria, (endo)lysins can also act as exolysins because the pepticoglycan layer of the bacterial cell wall is, in most cases, accessible from the outside. This is not the case for gram-negative bacteria, in which the presence of the outer membrane effectively prevents access by hydrophilic lytic enzymes.
However, when the lipopolysaccharide layer is disrupted (by EDTA,detergents, etc.) cells become sensitive to external murein hydrolases.

Lysin domains can be cloned into non-host bacteria and purified after expression and used for different applications.

Lysins have now been used successfully in animal models to control pathogenic antibiotic resistant bacteria found on mucosal surfaces and in blood. The advantage over antibiotics are their specificity for the pathogen without disturbing the normal flora, the low chance of bacterial resistance to lysins and their ability to kill colonizing pathogens on mucosal surfaces, capabilities that were previously unavailable. Thus, lysins could be an effective anti-infective in an age of mounting antibiotic resistance.
A potential concern in the use of lysins is the development of neutralizing antibodies. Unlike antibiotics, which are small molecules that are generally not immunogenic, enzymes are proteins that stimulate an immune response when delivered both mucosally or systemically. It was found that highly immune serum slows, but does not block, the killing of bacteria by lysins.

Similar to other proteins that are delivered intravenously to animals and humans, phage enzymes have a short half life (ca. 15 min.). However, the action of lysins for bacteria is so rapid, that this might be sufficient time to observe a therapeutic effect.

Because of the specific action of lysins, they offer a unique possibility for the biological control of unwanted bacteria without having any effect on other organisms. The most obvious approach to the use of lysins for the biocontrol of pathogens in food and feed is to directly add purified enzyme to the food or to the raw product. A more elegant and also less expensive alternative is the production and secretion of specific endolysins by fermenting. In this case the cell wall of the host bacterium must be insensitive to the produced lysin. By contrast, some applications aim to cause self-destruction, which is mediated by cells carrying endolysin genes that are able to degrade their own murein.

As bacteria get resistant to phages quite rapidly, they could also become resistant against lysins. However, repeated exposure of several bacteria grown on agar plates to low concentrations of lysins did not lead to the recovery of resistant strains. The cell wall receptor of lysins of S. pneumoniae is choline, a molecule that is essential for pneumococcal viability.
Although not yet proven, it is possible that during interaction of phage and bacteria over the millennia, to avoid becoming trapped inside the host, the binding domain of the lytic enzymes has evolved to target a unique and essential molecule in the cell wall, making resistance to these enzymes a rare event.

Differentiation of Bacteriophages on the basis of their Life Cycle

The differentiation between phages on the basis of their type of genetic material is a rather technical one. Another important differentiation can be made on the basis of the Phage Life Cycle.

Two main groups are distinguished there:

• Lysogenic or Temperate Phages

• Lytic Phages

Lysogenic phages

The most important feature of lysogenic phages is the integration of their genome in the bacterial genome after infection.
This results in the so-called pro-phage stage.

Genes that control the lytic pathway are repressed by protein repressors which bind to specific DNA-regions. This prevents
transcription of genes whose products are involved in the lytic pathway. During this stage the bacteria grow and divide normally and the phage inside the bacteria can remain unnoticed.

Upon activation of the pro-phage, in many cases for as yet unknown reasons, the bacteriophages genetic material is transcribed and replicated inside the bacterium. This ultimately results in
the formation of new bacteriophage particles which, once released form the bacterium, are capable of infecting new bacteria and integration of their genetic material into the bacterial genome, thus closing the circle.
Prophages are released by excision. Sometimes excision is not precise and bacterial genes adjacent to the prophage are packaged into a phage particle. These bacterial genes then can be transferred into other percipient bacteria and during, the formation of the prophage stage, integrated into the chromosome.

This process is called Transduction and has not
only led to the exchange of bacterial DNA between bacteria but also to the transfer of bacterial DNA to the genome of certain phages.
A nice example of this is the presence of so-called ‘pathogenicity islands’ in the genome of CTX Phi phage of the Vibrio cholerae (cholera bacterium) which contains a bacterial toxin encoding gene.

Although highly interesting, lysogenic phages are (as yet) unpredictable in their properties and behaviour. Transition from the pro-phage stage to the lytic phage is uncontrollable and the exchange of genetic material unpredictable.
Lysogenic phages have been observed to transfer potentially harmful genes into their target bacteria (including genes encoding Shiga toxins of Shigatoxinogenic E. coli , the cholera toxin of toxinogenic Vibrio cholerae, and various antibiotic resistance genes.

Although many of the lysogenic phages are relatively well studied (e.g. Phage λ) and sometimes detailed knowledge is available about their genome and its regulation, because of their unpredictable behaviour, they are generally considered to be unsuitable of use in bacteriophage therapy.

Lytic phages

Lytic phages infect their host, replicate and then kill their host by cell lysis, thereby releasing progeny viruses into the environment.

Infection of the bacterial host is often based on specific recognition of particular components (receptors) on the pili or cell wall of the bacterium which is likely to determine the specific recognition. After initial infection it can be a matter of hours before cell lysis occurs.

Lytic phages are theoretically ideally suited as an alternative antibiotic in phage therapy:

• Most bacteriophages are very specific for their host bacterium.

• Upon successful infection they replicate exponentially.

• They kill their hosts quickly (often in hours).

• Upon bacterial lysis a large number of progeny phages are released into the environment.Each of those phages is capable of infecting other target bacteria. As an ‘antibiotic’ bacteriophages are ‘self multiplying’.

• Bacteriophages depend on their hosts and will disappear with the destruction of their target bacterium leaving no residues; ‘antibiotic’bacteriophages are ‘self eliminating’.