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2007 Beeston, Paul S by wfq74180


									                          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

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