(125d) Mini-Bioreactors for the Study of Pneumococcal Cell-Cell Communication

Microfluidics allows for real-time single cell visualization and high-throughput experimentation. We have leveraged microfluidics to elucidate the relationship between signaling, population density and cellular heterogeneity in Streptococcus pneumoniae (Spn). Our long-term goal is to understand whether and how cell-cell communication in Spn orchestrates its lifestyle switch from a commensal to a pathogen. Our microdroplet-based technology platform consists of a highly monodisperse water-in-oil emulsion. We have successfully encapsulated Spn cells, and their signaling molecules, in these miniature bioreactors. Spn undergoes multiple rounds of division within the droplets, providing an optimal opportunity to quantify the association between cell-cell signaling, cell density, and environmental factors such as nutrient availability, pH, oxygen and temperature. We are investigating the kinetics of signaling of the TprA regulator and its cognate peptide PhrA; this signaling system promotes virulence in animal models of Spn carriage and pneumonia. To monitor PhrA signaling in real time, we utilize a reporter where the promoter of PhrA drives the far-red fluorescent protein, mCardinal. We have captured cell-density dependent PhrA signaling in a droplet at a single-cell level. We are currently quantifying the association between cell number and signaling, and its dependence on environmental conditions. Further, we are expanding this system to monitor multiple peptide-dependent signaling systems. In the long term, we hope to shed light on the relationship between the kinetics of Spn cell-cell signaling, environmental conditions, and virulence, and to exploit this knowledge for the development of anti-pneumococcal therapies.