Hello there! My name is Dustin Lee and this is my second blog post for the summer. Last time on Pace Undergraduate Research, I informed you that Dr. Chan and I had found out that there are amyloid positive sequences in Cryptosporidium parvum adhesion proteins. This assured us of the presence of amyloids in the parasite C.parvum. This is crucial because our project’s basis is on inhibiting amyloid formation in C.parvum’s adhesion proteins; thus reducing adhesion to the host HCT-8 cells. If the parasite can’t adhere to the host cells, infection is unfeasible.
We stained C.parvum for fluorescence using the amyloid-perturbing dye Thioflavin S (ThS). Staining for fluorescence helps us analyze the sporozoites’ biochemical and physiological activity. Using concentrations of ThS at 1 µM and 100 nM (0.1 µM), we took pictures of the stained sporozoites using fluorescent microscopy. Usually, one wants their picture to be in the highest resolution and as detailed as possible. Similarly, we wanted our fluorescence pictures to be visually clear and the features to be easily seen. The picture’s quality would affect our ability to examine the intensity of the fluorescent parasite. C.parvum sporozoites fluoresce better with ThS at a concentration of 1 µM.
We treated the parasite with other amyloid dyes as well. Thioflavin T (ThT), Thioflavin S, and Congo Red (CR) are the aromatic dyes we used to inhibit adhesion. The concentrations of the dyes were 10 µM and 200 µM of ThS, 1 µM and 20 µM of CR, and 0.1 µM and 2 µM of ThT. After allowing the treated parasites and non-treated C.parvum to infect the host cells through incubation, we used the Merifluor reagent to analyze adhesion. Merifluor is a reagent that uses direct immunofluorescence to detect/enumerate the C.parvum oocysts in a sample. With a known quantity of C.parvum oocysts, we are able to quantify the amount of sporozoites that adhered to the host HCT-8 cells. HCT-8 is the intestinal adenocarcinoma cell line that biochemically and physiologically mimics the epithelial tissue found in gastrointestinal areas like the small intestine. This cell line allows us to conduct in vitro research with C.parvum. With the data gathered, we noticed that the uninfected HCT-8 cells’ fluorescence intensity were measured at approximately 4000 Relative Fluorescence Units (RFU). The infected but non-treated host cells’ fluorescence was measured at about 7000 RFU, meaning there is a drastic increase in fluorescence when infected (nearly twofold). When treated with 10 µM of ThS, the infected HCT-8’s fluorescence was measured at a little over 6000 RFU, not showing a significant decrease in adhesion. With 200 µM of ThS, the infected HCT-8’s fluorescence rose even closer to that of the infected HCT-8’s level of fluorescence. 1 µM of CR reduce the infected HCT-8’s fluorescence to about 5000 RFU, but it still wasn’t a significant reduction. When treated with 20 µM of CR, the infected HCT-8’s fluorescence decreased to approximately 3500 RFU, a drastic decrease that is closer to the level of fluorescence of the non-infected HCT-8 cells. A treatment of 0.1 µM of ThT also significantly reduced adhesion to about 3800 RFU. Infected HCT-8 treated with 2 µM of ThT was measured at a fluorescence of 5500 RFU. The most effective treatments for reducing adhesion of Cryptosporidium parvum to HCT-8 were 20 µM of Congo Red and 0.1 µM of Thioflavin T.
Dr. Chan and I found that Cryptosporidium parvum sporozoites fluoresce best with 1 µM of ThS. We also discovered that 20 µM of Congo Red and 0.1 µM of Thioflavin T are the ideal concentrations and dyes to inhibit adhesion of C.parvum to the host HCT-8 cell line. In the future, we will play around with the concentrations of the aforementioned amyloid dyes in order to see if there is a dose-dependent response on fluorescence and adhesion.
Some challenges we encountered were precipitating a sufficient amount of C.parvum cells for staining/treating and taking pictures of the fluorescent sporozoites. Time can play a very crucial role when it comes to preparing a sample. e.g. When spinning down our C.parvum oocysts in a centrifuge in order to muster the parasites, our precipitate was occasionally smaller than expected; resulting in less cells than expected. This makes it more difficult to stain or treat the parasite based on the fact that there is less of the sample to work with. Speaking of staining, fluorescent microscopy also came with its challenges. Pictures didn’t always come out clear and we had to keep taking various photos until the pictures were more detailed. It also isn’t any easier when the sporozoites’ fluorescence intensity degrades within a certain window; hence there is a time frame when performing this type of fluorescent microscopy. The fact that we were able to bring down the level of adhesion back to 4000 RFU is fantastic. Honestly, I didn’t expect to have such efficacious results in this experiment. However, there is always room for improvement. This is why Dr. Chan and I plan to find a more ideal concentration of Congo Red and Thioflavin T to inhibit adhesion of C.parvum to the HCT-8 cells.
This pedagogic experience has taught me so much. I learned that results can have numerous interpretations, and that isn’t necessarily a bad thing. Additionally, methodology in an experiment may have to be altered in order to reach a desired result. Overall, I’ve learned a great deal over the course of this summer. A mentor-student relationship is something else. As I’ve stated before, if you are eager to learn in a specific field, then I highly recommend going under the tutelage of a mentor in that respective study. You will be shocked about how much you will learn, especially outside of the classroom. I thank Pace University’s Summer 2014 Undergraduate Research Program for this valuable opportunity.
Until next time,