Sathvik Bilakanti: Developing Nanobodies to Combat Parasites

What do you think of when you think of a human parasite? Most people might think of blood-sucking creatures like vampire bats and fleas, or maybe tapeworms. However, the vast majority of human parasites are much, much smaller. Diseases like malaria, dysentery, athlete’s foot are all caused by microscopic parasites. In fact, millions of people in the United States alone have parasitic infections, and most of them don’t even know it.

In Fall 2020, I started my UROP project in the Tomko lab at the FSU College of Medicine’s Department of Biomedical Sciences studying a little-known family of parasites known as microsporidia. These tiny parasites are most closely related to fungi, and live inside the cells of other organisms, including insects, fish, birds, and even in mammals such as humans. In some parts of the World, as much as 50% of the population may be infected with microsporidia, and although most people never know they are carrying these parasites around, others can develop lethal infections. Microsporidial infections are especially problematic among the immunocompromised, such as those who have received organ transplants, have inflammatory diseases such as rheumatoid arthritis, or who have HIV.

The Tomko lab has traditionally been interested in the proteasome, which is a recycling center for proteins that is found inside virtually every cell. By turning damaged, defective, or unneeded proteins back into amino acids, the proteasome provides the building blocks to make new proteins needed by cells to perform various functions. The proteasome in microsporidia is very different from the one found in people, and preliminary data from our group and others indicates that blocking the recycling of proteins by the microsporidial proteasome causes them to die.

It is our hope that we can develop inhibitors of the microsporidial proteasome as new antimicrobial agents to treat microsporidial infections in humans. A major roadblock to this goal is that at present, no tools to study, isolate, or track the microsporidial proteasome exist. In light of this, my IDEA grant will fund my work to develop nanobodies—small, genetically engineered antibodies—to track, purify, and manipulate the microsporidial proteasome for further study. For example, successfully purifying microsporidial proteasomes will allow us to search for drug candidates that block its function.

To develop microsporidial proteasome nanobodies, I cloned four different subunits of the microsporidial proteasome, and produced them in bacteria. I then used these recombinant subunits as bait to fish for nanobodies that would bind tightly to subunits of the microsporidial proteasome. The “pond” in which I went fishing was a yeast surface display library that consists of approximately 500 million yeast cells, each of which produces a different and unique nanobody on its cell surface. Using approaches called Magnetic Affinity Cell Sorting (MACS) and Fluorescence-Assisted Cell Sorting (FACS), I isolated the cells that have nanobodies that stick to the microsporidial proteasome subunit, and washed away all of the cells that did not. From this enriched pool of cells, we then isolated individual cells, and performed DNA sequencing to figure out exactly which nanobodies these cells produce.

For the coming summer, I will characterize a number of the nanobodies I discovered. An ideal nanobody will bind very tightly to the microsporidial proteasome, but won’t stick to the proteasomes of the infected human host cells, and will be able to bind to its target subunit under a variety of common experimental conditions in the lab. Once ideal nanobodies are identified our next goal will be to use them to purify the microsporidial proteasome from infected human cells so we can develop a better picture of how it is similar and different to that of humans, and to begin functional studies en route to drug screening. Importantly, by sharing these tools with other groups, we can help current and future scientists dive deeper into the mysteries of parasitic infections. The work supported by my IDEA Grant can potentially lead us to find a cure for these infections.

Besides conducting research, I am also involved in several organizations at FSU including WhoWePlayFor, which is an organization that promotes heart screenings and Alpha Epsilon Delta, which is a pre-health honor society.. These organizations helped me better myself as a person and understand the true value of service and helping those in need. My long-term career goal is to earn my Ph.D. and/or my M.D. and continue working at the interface of scientific research and human health. My IDEA Grant is giving me the experience I need to be confident in my decision, and the rigorous training to ensure I’m successful in my next step after FSU.

our next goal will be to use them to purify the microsporidial proteasome from infected human cells so we can develop a better picture of how it is similar and different to that of humans, and to begin functional studies en route to drug screening. Importantly, by sharing these tools with other groups, we can help current and future scientists dive deeper into the mysteries of parasitic infections. The work supported by my IDEA Grant can potentially lead us to find a cure for these infections.Besides conducting research, I am also involved in several organizations at FSU including WhoWePlayFor, which is an organization that promotes heart screenings and Alpha Epsilon Delta, which is a pre-health honor society.. These organizations helped me better myself as a person and understand the true value of service and helping those in need. My long-term career goal is to earn my Ph.D. and/or my M.D. and continue working at the interface of scientific research and human health. My IDEA Grant is giving me the experience I need to be confident in my decision, and the rigorous training to ensure I’m successful in my next step after FSU.

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