Logan Collins – TRANSCRIPT
Antibiotic resistance is an extremely serious threat in today’s world. It costs the US healthcare system over 20 billion dollars per year. And it kills an estimated 23,000 people per year in the US alone. In addition, there are concerns that antibiotic resistance might result in something far worse. It may result in something along the lines of the bubonic plague, which killed approximately a third of Europe’s population of the time that it occurred. We, of course, don’t want this.
So what’s being done about this? Well, traditional antibiotics tend to use compounds which will fit into the molecular machines that run bacteria, the macromolecules, those large molecules, that help to make bacteria work. And what they do is they’ll fit into a particular crevice as a key fits into a lock. And the antibiotics will inactivate these macromolecules and disrupt their function, thus killing off the bacteria. However, when bacteria in the population have variations of these molecules, which are differently shaped, the antibiotic key can no longer fit into the large molecular lock. And thus, the bacteria gains resistance.
Then these bacteria propagate in population, and it results in an antibiotic resistance population of bacteria. There are also some other ways that antibiotic resistance arises, including antibiotics not being able to enter the cell, antibiotics being pumped out of the cell, and enzymes which will chop up the antibiotics. I’ve been conducting research at the University of Colorado, Boulder, at the Chatterjee Lab, and thus far, I’ve gotten some very encouraging results, and I’m excitedly continuing to pursue the next steps in my research.
So what have I been doing? I have created an artificial gene that codes for antimicrobial polypeptides. This artificial gene is — Well, let me first explain what that means. Basically, I created a gene that is capable of synthesizing misfolded proteins upon entering into the bacterial cell. These misfolded proteins disrupt the biochemical balance in the bacterial cell by causing chaos through aggregation.
And I’ll get back to that in just a moment. As you can see, this causes a widespread disruption in the bacteria, rather than targeting a specific molecule. And because of this, it’s got great promise in being more effective than traditional antibiotics because the bacteria won’t be able to simply change the shape of one molecule. They will have to undergo a system’s wide adaptation if they are to gain resistance.
In designing this gene, I first wrote out an amino acid sequence. And then I converted it to a DNA sequence using software. In the design of this, I included several key elements. I included a promoter, an open reading frame, and a terminator. And the promoter is simply a piece of the DNA that is capable of activating the gene. That is, it’s what the bacteria are able to turn on once the gene has entered their cell. And this could be made specific to specific types of bacteria.
The terminator turns off the gene once it’s finished being transcribed. It limits it to just the specific sequence that is to be translated eventually. And the open reading frame contains the actual information which will go into the sequence of the gene, or the polypeptide, that is. When designing this gene, I specifically included a lot of hydrophobic amino acids. And the reason for this was because I was trying to vastly increase the likelihood that aggregates would form in the bacterial cells.
As I’ve mentioned earlier, aggregates can be toxic and cause chaos through a variety of mechanisms. The reason that hydrophobic polypeptides tend to aggregate is because they clump together in the presence of water. They are afraid of water. It’s more thermodynamically favorable for them to avoid water. So, by clumping together, they minimize the exposure of their surface to the water in the bacterial cell.
This also provides potential benefit in that the aggregates most likely would be much more difficult to remove from the bacterial cell as traditional antibiotics are when they are pumped out. Another benefit of having very hydrophobic polypeptides is that they may take on multiple shapes. This is because the polypeptides wouldn’t necessarily just have one way of folding because they’d always be exposing hydrophobic patches. They’re so hydrophobic there’s no way for them to hide all of the hydrophobic bits. And so, they would have to, depending on the environment in the cell, be re-folding themselves to maximize the amount of the surface area that’s being hidden from the water.
And because of this, it would possibly be more difficult for bacteria to recognize specific shapes of the polypeptides to chop up with enzymes. To deliver this, I’ve decided that bacterial conjugation is an ideal delivery system, and I’ve begun to work on this. And as I’ve mentioned, I’ve gotten some very promising results with it. So bacterial conjugation is the transfer of DNA from one bacterium to another. It’s essentially bacterial sex.
And because of this, it’s another potential barrier to antibiotic resistance because bacteria have been using this to their advantage for millions of years. And as a result, to remove their ability to conjugate, would be a selective disadvantage because it would remove a beneficial adaptation. Again, a barrier to antibiotic resistance.
Going back to the actual delivery, the way that this would work, the donor bacterium will transfer a plasmid, which is a circular piece of DNA, in general, and the plasmid would contain the artificial gene. And then, once the donor transfers the plasmid to the recipient, it enters the recipient and activates the artificial gene based on a pathogen specific promoter.
That is a promoter that only a certain type of bacteria, in any pathogen you choose to target, would be able to activate. And then, the recipient pathogen would be in to produce the anti-microbial polypeptide, and it would kill off the recipient. To accomplish this, I chose the RK2 plasmid, which is a broad host range conjugative plasmid. That is a plasmid, as I mentioned, a circular piece of DNA that is capable of synthesizing all the necessary proteins to transfer itself between bacteria. It can transfer itself between many different types of bacteria so it can transfer itself exponentially, going from a small group of bacteria to several more and several more, etc, until it spreads throughout a population.
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