Quantitative analysis of bacterial cell-cell communication at the single-cell level using microdroplet arrays.

Cell-cell communication (CCC) contributes to bacterial survival and adaptability. Gram-positive bacteria employ secreted peptides to coordinate CCC. While the molecular pathways activated by these peptides are well studied, little is known about how individual cells contribute to initiating the signaling response. To address this question, we used microdroplet arrays to examine the major human pathogen Streptococcus pneumoniae and its TprA/PhrA regulator/peptide CCC system, which promotes colonization and virulence. We measured phrA promoter activity in wild-type (WT) cells and in a phrA deletion mutant, using populations seeded before signaling began. As signaling emerged, we observed heterogeneity in S. pneumoniae signaling within and across microdroplets. Addition of exogenous PhrA increased both the magnitude of signal and the percentage of signaling cells, yet it did not reduce the heterogeneity of signal. When examining whether PhrA peptide produced from WT cells was shared with ΔphrA cells, we found a preference for self-signaling over signaling to neighboring cells. Overall, we developed a platform to quantify cell-cell signaling at the single-cell level and determined that at early stages TprA/PhrA signaling is highly heterogeneous and primarily targets producing cells. We propose that this heterogeneity and its amplification through autoinduction may confer a fitness advantage to the population.