Crithidia fasciculata is a protozoan parasite that is present in the environment and infects mosquitoes. Ingested parasites travel through the mosquito digestive tract and adhere to epithelial cells of the hindgut. C. fasciculata rosettes, adherent parasites replicating in flower-like clusters, can be grown in culture and structurally resemble parasites present in the mosquito hindgut. The main goal of this project is to understand the adherence mechanism utilized by this parasite in mosquitoes. By studying C. fasciculata, we hope to use it as a model organism more broadly for the insect stage of trypanosomatid disease transmission. This includes human pathogenic organisms such as Leishmania and Trypanosoma which combined affect over eight million people yearly.
This summer under the mentoring of both Michael and Megan Povelones I made progress in studying both the cell and molecular biology aspects of the project. One of the central aspects of the project is targeting and disabling genes which were gathered from RNA sequencing (RNAseq) analysis of both adherent and swimming forms of C. fasciculata. I learned how to use a four way ligation strategy involving a drug plasmid, and upstream and downstream homology arms in order to create a construct able to knockout the gene of interest. Once both alleles are knocked out the mutant C. fasciculata will be observed for any phenotypic changes. I was able to successfully create six constructs to target three different genes and am now in the process of transfecting them into the parasites.
The relative slowness of having to individually knockout both alleles of a gene of interest is inhibitive to effective study of the large number of genes generated from the RNAseq analysis. As result, we have begun the process of modifying and implementing a higher throughput system of knockout involving a CRISPR-Cas 9 protocol designed for Leishmania. There is a large amount of information to learn on my end for this new project design, but it would allow me to target both alleles simultaneously, saving time which can be used to target more genes.
Also, this summer I created a Shiny app using R to explore the RNAseq data generated from our earlier study. I also did a pair of live imaging studies observing over two days how C. fasciculata adhere to a well, grow, and divide. This was a very interesting study and has led to many research questions about the dynamics of swimming and adherent cells.
Lastly, this summer I did quantitative PCR to look at mosquito immune levels over multiple days of C. fasciculata infection. We knew from the RNAseq data that there was potentially some immune activation by the mosquito in response to parasite infection however this had not been validated. We saw in each replicate at day 7 or 14 a small dip followed by a massive peak in the number of copies of RNA for the gene Defensin A (Def A). Def A is an antimicrobial peptide involved in the innate immune response of mosquitoes.
My experience in the Povelones Lab has provided an incredible look at the inner workings of biomedical research. It was amazing to be able to apply information I learned in my classes to help plan, execute, and analyze my own experiments. I look forward to learning more laboratory skills, and information in the upcoming semesters. I would like to thank everyone in the Povelones Lab, CURF, the College Alumni Research Society, and the College House Research Fellowship for continuing to provide support and make this possible.