Systematic identification of interactions between Lyme Borrelia and Ixodes scapularis - PROJECT SUMMARY This proposal aims to investigate the interactions between Lyme disease bacteria, Borrelia burgdorferi, and extracellular, secreted and transmembrane proteins (exoproteins) of its common North American tick vector, Ixodes scapularis. This is an extension of our previous work leveraging a yeast display library comprising over 3,000 human exoproteins to systematically profile human-Borrelia interactions (Cell 2024 Bibliography reference 1) 1. These human studies identified numerous interactions important for Borrelia infection, such as PGLYRP1 with Lyme Borrelia 3 and CD55 4 with relapsing fever Borrelia. Now, we have developed a similar library termed IscREAM (I. scapularis Rapid Extracellular Antigen Monitoring), of over 3,000 carefully curated exoproteins of I. scapularis. We propose to screen this library with diverse strains of Lyme Borrelia, and validate top hits by ELISA and flow cytometry. Lyme Borrelia interactions with Ixodes exoproteins are critical nodes facilitating pathogen colonization of the vector, persistence within the tick, and transmission to vertebrate hosts. Better understanding of these interactions is important for the development of strategies to block transmission or interrupt the disease cycle. Unfortunately, traditional methods to identify these interactions have been limited in scale. For example, ELISAs and flow cytometry have been impeded by their experimental throughput, while high throughput methods such as mRNA capture libraries have been constrained by library quality. IscREAM, on the other hand, mitigates many of the inherent shortcomings of traditional methodologies through its careful curation and high throughput nature. IscREAM is comprised of barcoded yeast clones expressing 3,177 individually selected and transformed exoproteins. This library has been validated with sera of guinea pigs vaccinated against specific I. scapularis antigens, and we found IscREAM performs with a high degree of sensitivity and specificity. Now, we propose to mirror our previous human library screens 1,3,4, and probe IscREAM with diverse strains of Lyme Borrelia. To do so, the IscREAM library will be probed with biotinylated spirochetes, and spirochete-bound yeast clones will be isolated via magnetic separation with streptavidin coated magnetic beads. Borrelia-binding IscREAM proteins produced on the surface of the yeast will then be identified by next generation sequencing of protein-specific genetic barcodes. We will validate top hits by ELISA and flow cytometry. This effort will yield a database of interactions between Lyme Borrelia and I. scapularis exoproteins. This will foster further research into potential vaccine targets and prophylactics to impede tick-to-host transmission of Lyme Borrelia, advancing our ability to combat Lyme disease effectively.
