(759h) Demonstrating Cell-Communication in Droplets to Target Novel Antimicrobials for Streptococcal Infection

Although Streptococcus pneumoniae (Spn) is a major human pathogen that causes over a million annual deaths in young children and the elderly, worldwide, it also colonizes the human nasopharynx of many humans without causing any symptoms in its commensal form. After Spn colonizes the upper respiratory tract (URT), it can travel to other tissues in the host, causing mild or severe diseases, and increased virulence. Many questions remain about what causes Spn cells to switch from being a commensal to a pathogenic strain, like what molecules coordinate these processes, and how does cell density influence virulence. Our overarching goal is to understand how population-level influences Spn communication and how communication may orchestrate this life style switch. To answer these questions, we have leveraged a droplet-based technology platform that offers a two-pronged approach when determining how cell density and communication enables us to investigate virulence in humans, and when testing whether an inhibiting strain in other bacterial cells in the URT can kill Spn. By encapsulating Spn strains and signaling molecules in microdroplets, a highly-uniform water-in-oil emulsion that act as miniature bioreactors, we can monitor cell expression in cells in real time. Our lab has reported a specific set of genes in Spn that are associated with increased virulence in mice that is known as the SHP144/Rgg144 transduction system. Changes in the expression levels of these genes was previously monitored using a lacZ assay. However, in droplets, we will replace the lacZ gene with a red-orange fluorescent protein, tdTomato, such that modulation of the SHP144 gene can be monitored in real-time using fluorescence. Our first aim will validate microdroplets as a proof-of-concept that demonstrates cell-communication in Spn. We will do this by removing the SHP144 gene from the Spn wild type (WT), which is called a SHP144 mutant, and add exogenous SHP144 molecules to determine whether these molecules can induce SHP144 expression in the SHP144 mutant in droplets. We will also co-encapsulate a SHP144 mutant with a WT cell to determine whether the WT can induce signaling in the SHP144 mutant. We will determine how the initial number of cells in droplets influence the optimal conditions for SHP144 expression. Our second aim will identify and isolate target molecules from a large number of peptides that will kill the WT Spn cell. We will do this by developing a label-free unique color-coded identifier where each molecule or peptide, is mixed with a unique mixture of quantum dots (QDs) by mixing different ratios of yellow and red QDs to make different colors. These unique mixtures of QDs will assigned to and mixed with a respective inhibiting peptide that was selected from previous work, and a gel to form tiny beads of different colors that each correspond to a different inhibiting peptide. Each colored bead will be injected into droplets that contain the WT Spn cell. As the droplets incubate, we predict that the inhibiting peptides will reduce the signal in the WT Spn, which we would characterize as an inhibiting molecule. Molecules that reduced the tdTomato expression will be classified as an inhibitor and identified based on color. Future work enables the colored beads and droplets to be sorted using a flow cytometer with 16 different colors and a four-way sorter QDs synchronously emit different colors, or with a droplet sorter that we build in our lab. Together, results from our findings will uncover the contribution of cell density, to the Rgg144/SHP144 system, of the Rgg144/SHP144 system to host virulence, our understanding of pneumococcal colonization, and shed light on the development of anti-pneumococcal therapies. Moreover, it will provide a means to elucidate peptide-mediated cell signaling in a high-parallel manner, and will be readily adaptable for high-throughput screening of libraries downstream.