In today’s Student Research Spotlight, a predental member explains her research into Gonorrheal DNA enzymes.
Note: “LtgA” refers to the protein, while “ltgA” refers to the gene itself
Neisseria gonorrhoeae are gram-negative bacteria that can be transmitted sexually or during childbirth, leading to infections of the genitals, rectum and throat. Although gonorrhea is a treatable disease, many people are asymptomatic and go undiagnosed. If left untreated, gonorrhea can result in infertility. Recently, the development of antibiotic resistant strains has become an increasing public health concern. Research teams are focused on learning more about the bacteria so that new antibiotics can be developed.
The cell wall of gonorrhea, which is composed of peptidoglycan, is a major virulence factor of the bacteria and a target for medications.
Gonorrheal DNA encodes for enzymes known as lytic transglycosylases (Ltg), which are able to cleave peptidoglycan into monomers and release them.
This process furthers damage to the host. My research project is centered on a specific Ltg, LtgA. In a recent study, it was reported that when ltgA was mutated, there was a decrease in peptidoglycan monomers being released. Medications that regulate the production of LtgA could be used to treat gonorrhea.
Although there is plethora ways in which genes can be regulated, one of the major ways is via antisense RNA. Antisense RNA is merely a short chain of RNA that can bind to single-stranded RNA. When antisense RNA binds, it can create double stranded RNA that is degraded, or open up or create RNA stem loop structures.
All these scenarios can affect protein production. There are 253 known anti-sense transcripts present in gonorrhea, and one of them binds to ltgA. Therefore, the focus of my research is to determine if –and how—the antisense transcript change the expression level of LtgA.
The first step in answering this question required the construction of new plasmids to amplify the region of DNA that encoded for the antisense. I inserted the antisense region of DNA into vectors, and then transformed the plasmids into E. coli cells. Then, the amplified plasmids were cut with an enzyme and transformed into gonorrheal cells. Using this method, six new gonorrheal strains containing ltgA were created. Next, some of the strains were induced to transcribe the antisense, and a Western Blot Analysis was performed to measure the production of LtgA.
In the end, the results were not significant. However, there did seem to be a trend where an increase in antisense caused a decrease in LtgA production. The next step in this project will be to perform a Northern Blot Analysis, which will confirm that the antisense RNA is even being transcribed.
~Sara DeGrave, UW-Madison ’16, predental member