Beeston, Paul Henry The Role of YPO 2169 in Flea Transmission of Plague Faculty Mentor: David Erickson, Microbiology and Molecular Biology Yersinia pestis is the gram negative bacteria responsible for the disease known as the plague. It is transmitted to hosts primarily through fleas such as the Oriental rat flea North American ground squirrel flea. Y. pestis is mainly a disease that affects small mammal populations such as rats and squirrels. The environment of the rat flea and other fleas, however, is very different from small mammal hosts or human hosts. Fleas maintain a body temperature considerably lower than mammals. Fleas also have unique physiologies that greatly differ from mammals. When Y. pestis infects the rat flea it multiplies and forms a sticky biofilm in a valve between the esophagus and the midgut called the proventriculus. After the adherent biofilm has built up enough to completely block the digestive tract of the flea, the flea will increase the number of feeding attempts. As it attempts to feed on a mammal, bacteria can become dislodged or regurgitated into the host, thus spreading the disease. In order to complete this process, Y. pestis must combat the flea immune system and grow without killing the flea so it can be transmitted. Very little is known about how Y. pestis does this. Although the Y. pestis genome has been completely sequenced, much is still unknown about how each gene works and affects the bacteria’s ability to infect. At best we can guess or make predictions of which genes do what based on their similarities to known genes. One Y. pestis gene in particular, YPO2169, has similar gene sequences to genes in a group of bacteria that are known to infect insects. This is a good indicator that this gene may play a significant role in Y. pestis’ pathway of infection in the flea. The gene YPO2169 is homologous to the gene ytxR in a closely related bacteria Yersinia enterocolitica. It is proposed that the ytxR gene regulates certain toxins, but ytxR is conserved in many Yersinia species (such as Y. pestis) that do not contain those toxins, thus suggesting another function for that gene. I hypothesized that the YPO2169 gene regulates Y. pestis genes that are involved in flea infections. To study the effects of the YPO2169 gene I needed to compare genetically complete wild-type bacteria, with bacteria that had the YPO2169 gene removed. Removing a single gene while keeping all other genetic material intact is easier said than done. I used a technique called “lambda-red recombinase,” which has been used to knock out specific genes of E. coli. First I shocked samples of Y. pestis which stimulated them to uptake circular pieces of genetic information or DNA called plasmids. The plasmid contains a gene that increases the amount of homologous recombination or crossing-over that the genes do to vary their content. I then used a polymerase chain reaction (PCR) that uses the raw pieces of nucleotide to construct DNA strands. I constructed a strand of DNA containing a gene for resistance to a certain antibiotic and flanked the gene with sequences matching the sequences on the sides of the YPO2169 gene, thus when they recombine, the antibiotic gene would replace the YPO2169. I also had a marker to select which bacteria this switch happened in by growing the bacteria on plates with the antibiotic so only those that switched could survive. This process in long and time-consuming and after several unsuccessful attempts over two months, we asked a professor from Idaho who had success with this technique before on other genes to try it on our gene of interest and it worked. The mutant Y. pestis strain we called Y. pestis ΔytxR. We used an artificial feeder that stimulated fleas to bite through a mouse skin and ingest blood mixed with either wild-type or our mutant strain bacteria. I isolated the fleas that had been infected and observed twice a week how many died and how many had the proventriculus blocked from the biofilm. I also took samples of fleas at differing time periods and crushed them and plated them out to count the number of bacteria present in the fleas during differing times in the course of infection. The results we not as I had anticipated. I thought that the biofilm blockage might not be influenced but that without the YPO2169 gene, the bacteria would at least be less deadly or virulent to the fleas. Our data showed quite the opposite with 41 out of 100 infected fleas becoming blocked in the wild-type, and only 14 of the mutant-infected fleas being blocked. Notwithstanding the lower blockage rate, the mutant-infected fleas had more deaths than the wild-type (27 compared with 15). Multiple trials were done and yielded similar results to show that the Y. pestis ΔytxR strain killed more fleas than the original wild-type strain. What accounts for this difference? The gene could possibly code for a repressor in the bacteria that lessens its ability to infect. The gene could also collaborate with other genes in its functions. Though not thoroughly conclusive, this study provides valuable information into the function of a relatively unknown gene and its effects on the flea vector. This project can be followed up by others to further our understanding of this gene. I already have started some studies with nematode infections and surely others in the lab will continue. This project has been invaluable to me in giving me research laboratory experience and helping me apply so many concepts that I have learned in books to actual lab work that contributes to knowledge in the scientific community. I plan to continue my education in pursuit of a PhD or MD degree. Regardless of which road I chose, there will be many opportunities for me to continue to do research in the future.
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