Poster U19 - MINI-BIOREACTORS FOR THE STUDY OF PNEUMOCOCCAL CELL-CELL COMMUNICATION

Introduction: Our overarching goal is to understand whether and how S. pneumoniae (Spn) cell-cell communication orchestrates the switch from commensalism to pathogenesis. Spn is a frequent commensal that asymptomatically colonizes the nasopharynx in humans causes pneumoniae, sepsis, and meningitis in human. Here we focus on TprA/PhrA, a cell-cell communication system that promotes Spn virulence.

Materials and

Methods: We have leveraged a droplet-based technology platform that co-encapsulates Spn cells and their signaling molecules. As Spn undergoes multiple rounds of division within the droplets, it provides an optimal opportunity to quantify the association between cell-cell signaling, cell density, and environmental factors. We are investigating the kinetics of signaling of the TprA regulator and its cognate peptide PhrA. Using WT cells and a phrA gene deletion mutant, we monitor PhrA signaling by utilizing a reporter where the promoter of PhrA drives the fluorescent protein, mCardinal.

Results, Conclusions, and Discussions: We have established Spn growth in droplets and captured PhrA cell-signaling. Using confocal microscopy and Imaris we quantify the signaling of individual cocci, allowing measurement of signaling per droplet as well as signaling per cell within a droplet. These data reveal an association between signaling and the availability of exogenous peptide, as well as heterogeneity in the signaling of individual cells within and across droplets. We also assess interactions between individual cells and neighboring droplets using a multi effects model. Overall, we have developed a microfluidic platform with high-throughput and single cell capabilities to study bacterial cell-cell communication. In the long term, we aim to shed light on the relationship between the kinetics of cell-cell signaling and environmental conditions and to exploit this knowledge for the development of anti-pneumococcal therapies.

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