Thursday, April 21, 2011

Getting a grip on prophage

by Dr. Ray Nims

In a previous post, we discussed bacteriophage as a risk for the manufacture of biopharmaceuticals by bacterial fermentation. We mentioned briefly that bacteriophage may integrate within the genome of bacterial cells and that this may also represent a problem. Now we will explain why.

Bacteriophage are viruses that infect bacteria, and they have evolved two mutually exclusive strategies for survival. One involves a lytic growth cycle leading to death (lysis) of the host cell and release of progeny phage that may then infect additional host cells (so-called horizontal transmission). The other strategy is called lysogeny and involves integration of phage coding sequences into the host (bacterial) cell genome. The integrated phage is termed a prophage. This strategy for phage survival is referred to as vertical transmission since the phage genomic material is reproduced along with that of the host cell as the latter proliferates. Under certain circumstances, however, the integrated prophage can excise itself from the host cell chromosome in a process referred to as induction. The excised phage then may initiate a lytic infection of the host cell, causing all of the problems discussed in the previous post.

Illustration of a T4 phage infecting E. coli by Jonathan Heras

The relative success (i.e., from the perspective of the phage!) of the lytic vs. lysogenic survival strategies changes with the probability of host cell survival. Lysogeny appears to be a strategy that allows phage to persist during periods of low host cell availability or poor environmental (e.g., nutrient) conditions. Induction of prophage is an adaptation of the phage to host cell damage. This damage usually takes the form of a major stress to the host cell.

If stess can lead to prophage induction, the worry then becomes that some manipulation of a bacterial production cell during biopharmaceutical manufacture could lead to induction and initiation of a lytic phage infection. How can we assess and mitigate the potential for this to occur? There are two approaches: first, we can perform chemical or physical induction studies to determine the likelihood of encountering a prophage in a given production cell; and second, we can engineer the conditions of bacterial growth such that induction of a prophage is discouraged.

Phage induction studies may be performed on the bacterial production cell following initial engineering of the cell or during characterization of the cell bank. The inducing agent most often employed is mitomycin C. Other types of inducing agents (conditions) include carcinogens (such as the N-nitrosamines), hydrogen peroxide, high temperature, starvation, and UV radiation. The cells are treated with the inducing agent or condition, then one of various endpoints is used to detect the initiation of a lytic phage infection. These could include culture assays as well as molecular techniques such as PCR, microarray, or DNA chips.

Suppose you have an E. coli production cell harboring a problematic prophage. What can be done to discourage phage induction? Certain growth procedures have been shown to reduce spontaneous phage induction in E. coli cultures. These include using lower bacterial growth rates, replacement of glucose in growth medium with glycerol, and engineering the production cell through introduction of a plasmid conferring over-expression of the phage cI gene.

In summary, there are approaches that can identify the likelihood of encountering prophage induction from a bacterial production cell. The time to perform this type of testing is during development of the fermentation process (following the engineering of the production cell), or following banking of the production cell. If prophage induction appears to be a problem, bacterial growth procedures can help to reduce the potential. If this is not sufficient, the production cell may need to be re-engineered to produce a phage-resistant mutant.

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