Kody Johnson and Dr. David Erickson, Department of Microbiology and Molecular Biology
Among many transmissible infectious diseases falls the Bubonic Plague, a disease that is caused by Yersinia pestis, a bacterium. It is still a concern because there have been human cases as recent as last year in our home state of Utah. While health care has advanced significantly since the last large plague epidemic, there are still areas of the world where health care is not as efficient or advanced and it is in these areas that this disease causes the greatest harm.
Because Yersinia pestis is a vector-borne disease, there are many aspects of its life-cycle and host interaction that must be studied. One stage that I was interested in particularly was its ability to infect fleas prior to infecting humans. In order to infect people, the bacterium must first successfully colonize and block a flea’s proventriculus, causing the flea to starve and frantically attempt to feed. It requires only 5-10 bacterium to infect and eventually kill a human being but over 1000 to successfully infect a flea. The cause for this great disparity is unknown but is suspected to be partially due to the effective innate immune system of the fleas.
In working with my mentor, we discussed some research done by another group and settled upon a novel immune protein that had characteristics similar to a family of proteins in the human body called defensins. We hoped to focus on one most likely cause to narrow our possibilities initially. Our first step then was to create a copy of the defensin gene from the flea so that we could produce a large quantity of the protein. I did so by making primers specific for that gene and then performing a reaction that will amplify the amount of DNA of my choosing. Once I had a large number of copies of this gene I took another segment called a plasmid which is used to place a new gene into a bacterium, and spliced the two pieces together. I did this because one of the easiest ways to produce large quantities of a specific protein is to create a bacterial system that will express or make the protein for us. I took the gene copy and placed it into a strain of bacteria.
This method allows us to have a system that we can now control the expression or production of our protein of choice in theory. The problem lies then in practice. Many proteins that are not naturally produced by a strain may interfere with the growth of that type of bacteria. This peptide that we were trying to produce also has one other major disadvantage in a bacterial system: the protein’s function is to kill bacteria. The exact function or mechanism of action for this protein is unknown so we didn’t know whether the bacteria producing the protein would limit its effects because it would be inside and wouldn’t be able to affect the membrane or not.
There is one other special feature of the system that we used that was important for us and that is the presence of a control feature. There are certain sequences that will only respond when certain chemicals are present and if they are not, then nothing will happen. This was the case for us. Due to the toxic nature of this peptide, we had to try to let the bacteria grow without it for as long as possible. Once they had reached a sufficient number we would be able to cause them to start producing it or induce them and hopefully get enough protein to test. We grew the bacteria and then induced them. After breaking open the cells we had to purify the protein from the rest of the material present. When we used the plasmid to introduce the defensin gene into the bacteria, we had placed a special sequence at the beginning which would make the protein stick to nickel. This allows us to mix the broken up cells with small pieces of nickel and then wash away everything else. This also works in theory.
After doing this, we can test for the presence of protein by suspending the solution in a gel matrix and then adding a dye that binds to protein. We also have a marker that lets us measure the size of the proteins that are present. After doing all of this we did not have anything that was the correct size so our system did not work. I tried a couple of times thinking that maybe I had incorrectly performed one step but each time produced the same results so I had to switch systems, using a different plasmid and different tag. Each time I had success with the early steps but the final production of the protein was not working.
Because there are many different states that a protein can exist we needed to test different chemical conditions to try and bring the protein out. This was done also without success. Our thought is that the protein may be secreted and lost and so that is why we have not been able to recover any during our tests. To overcome this we have selected a new system that will create a double protein, sticking a protein that is known to go to one area in the cell, with our protein. This will help us to isolate the protein more effectively as we know exactly where it will be located and the protein binds to a very specific substrate, allowing for yet more accurate purification.
We have just begun work with this system and will continue to move forward hoping to isolate some defensin protein. As we continue to test out this new system we hope to be able to take the protein that we isolate and use it to conduct tests on the Yersinia pestis bacteria. In this manner we hope to isolate one major player in the life cycle of this bacteria.
Our next step will be to knock down or remove the gene for this protein from the flea to see if the bacteria are more or less able to survive and colonize. There may be other proteins involved and we will find out with continued research. This disease may be better managed and we will determine that with our findings.