Author : Adenosine.

Bacterial conjugation is the ability for one bacteria to transfer genetic material to another via a physical bridge between the cells. It is the only cell-to-cell contact method of horizontal gene transfer amongst bacteria. A separate, non-chromosomal DNA ring, known as an F-plasmid, is separated into two strands, and one of them transferred to the recipient bacteria. Both strands are replicated by DNA polymerases, resulting in both bacteria having a copy of the F-plasmid (Griffiths, 2000). Conjugation is used in nature to share beneficial genetic material between bacteria, such as antibiotic resistance. However, manually inserting genes into the F-plasmid would allow for scientists to have bacteria transfer almost any gene to other cells, including our AMP kill switch.

This could potentially give rise to what we see as a “bacterial anti-bacteria” system. By creating cells that can conjugate the kill switch-containing plasmid into non-host microbes, we could then activate that kill switch, disrupting the membranes of, and ultimately killing, those cells. Protegrin-1 also displays antimicrobial activity towards fungi as well, meaning that two separate infection types that would normally require different treatments could be dealt with via a single intercellular technique (Kokryakov, 1993).

Utilizing conjugation to pass this kill switch between cells has many benefits. These bacteria would be another weapon in the body’s arsenal to fight off infectious, invading microbes. All the genes required for conjugation to occur are found within the F-plasmid, meaning that once a non-host cell is conjugated with and receives the F-plasmid, it too can further conjugate the kill switch into other microbes (Griffiths, 2000). Not only that, but when the bacteria replicates within the body, the resulting twin cell will receive of copy of the original’s DNA, including the F-plasmid, meaning that the twin has both the kill switch present, as well as the ability to conjugate further. Once spread to multiple non-host microbes, the kill switch promoter could then be induced by outside means, such as the addition of a molecule into the body (resulting in mass non-host cell death), or induced statically within the body by a microbial activity to which the promoter is sensitive.

It is important to note that the trigger for kill switch activation could be designed before the kill switch is conjugated, and this could be tailored to the specifics of the situation at hand. Knowing the type of invading bacteria or fungi, including their preferred ecological niche or intracellular chemical pathways, could allow the promoter of the kill switch to be induced by a process specific to that microbe, removing the need for an outside promoter inducer. This would allow the kill switch to be more specific to the microbe, resulting in a better treatment of that infection.

Another benefit of using antimicrobial peptides over antibiotics to attack non-host bacteria reduces the chance of creating antibiotic resistance. The small-scale DNA mutations that occur in order for bacteria to resist antibiotics are nothing compared to the grand-scale mutations needed to completely and functionally re-structure a bacterial membrane. Antibiotic-resistant bacteria are incredibly dangerous, such as methicillin-resistant Staphylococcus aureus (MRSA) which causes upwards of 18,000 deaths per year in the U.S.A alone (Boyles, 2007). The treatment of these bacteria with more aggressive antibiotics only leads to higher and more complicated levels of resistance. Circumventing this process may actually save lives by reducing the chance of creating another antibiotic-resistant strain.

Conjugation presents many dangers as well. Because the process of in vivo bacterial conjugation cannot be entirely controlled by scientists, accidental horizontal gene transfer is a cause for concern. If the AMP gene were transferred to natural bacterial flora, they too would be damaged upon promoter activation. How exactly to prevent this from happening is unclear, as the use of conjugation as a method of DNA transfer, while well versed outside the body, has little research pertaining to its induction in vivo. Another problem with conjugation is the release of endotoxins from Gram-negative bacterial membranes upon disruption. Endotoxins cause an anti-inflammatory response from immune cells like macrophages or leukocytes, causing dangerous diseases such as meningitis or meningococcal infections. These conditions may well be more dangerous than the original infection we would be using conjugation to eradicate, and this is an important factor in the medical application of this kill switch.

Experiments concerning the nature of conjugation have been conducted since the early 1900s, but few, if any, were within a synthetic biology context. Now that the technology exists to create customizable DNA devices, the ability to move predetermined sequences of DNA from one bacterial cell to another is an untapped reservoir of potential scientific advance. Our kill switch, combined with conjugation, would give genetically engineered microbes the ability to induce scientifically-controllable, mediated cell death in other bacteria. The ability to do this has a wealth of applications both inside the body and out, and we believe it is an important and untapped resource for science to explore in the near future.

References:

Boyles, Salynn. "More U.S. Deaths from MRSA than from AIDS." WebMD Health News. Published October 2007. Link to paper.

Griffiths AFJ, Miller JH, Suzuki DT et al. "An Introduction to Genetic Analysis." 7th ed., 2000. Link to paper.

Kokryakov, Vladimir et al. "Protegrins: leukocyte antimicrobial peptides that Combine Features of Corticostatic Defensins and Tachyplesins." Federation of European Biochemical Societies, Vol. 327, No. 2, pg. 231-236. Published 1993. Link to paper.