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    Nano and Micro Technologies

    (I-354) Mini-Bioreactors for the Study of Pneumococcal Cell-Cell Communication

    Friday, October 13, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row I - Poster # 354

      Presenting Author(s)

    • Corine Jackman, PhD (she/her/hers)

      Presidential Postdoctoral Fellow
      Carnegie Mellon University
      Pittsburgh, Pennsylvania, United States

      • Co-Author(s)

    • AL

      Anne Liu

      Undergraduate in the Department of Psychology
      Carnegie Mellon University, United States

    • MH

      Matthew Hou

      Undergraduate in the Department of Biological Sciences
      Carnegie Mellon University, United States

    • LE

      Lydia Eutsey, Ph.D.

      Imaging Director of the MCS Shared Microscopy and Imaging Center
      Carnegie Mellon University, United States

    • FL

      Frederick Lanni, Ph.D.

      Associate Professor in the Department of Biological Sciences
      Carnegie Mellon University, United States

    • SA

      Shelley Anna, Ph.D.

      Prof. of Chemical Engineering, Associate Dean of Graduate & Faculty Affairs & Strategic Initiatives
      Carnegie Mellon University, United States

      • Last Author(s)

    • LH

      Luisa N. Hiller, Ph.D.

      Associate Professor in the Department of Biological Sciences
      Carnegie Mellon University, United States

    Introduction: Streptococcus pneumoniae (Spn) is a major human pathogen that causes over a million annual deaths in young children and the elderly worldwide. Spn can also colonize the human nasopharynx without causing any symptoms. Disease develops when Spn disseminates from the nasopharynx to other host tissues. Many questions remain regarding the molecular mechanisms associated with the switch from commensalism to pathogenesis. Our overarching goal is to understand whether and how Spn cell-cell communication orchestrates this lifestyle switch.
    Materials and Methods:

    To answer this question, we have leveraged a droplet-based technology platform that co-encapsulates Spn cells and their signaling molecules. Using WT cells and a phrA gene deletion mutant, we monitor PhrA signaling in real time by utilizing a reporter where the promoter of PhrA drives the far-red fluorescent protein, mCardinal.

    Results, Conclusions, and Discussions: 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. This signaling system promotes virulence in animal models of Spn carriage and pneumonia. We have measured cell growth and cell-signaling of individual cocci within each droplet. We captured phrA-dependent signaling in the WT by establishing a threshold where signaling of individual WT cells are on or off based on basal expression of the mutant for each experiment. These data have revealed a correlation between increasing cell-density and PhrA-dependent signaling across cells in droplets. We have also observed substantial heterogeneity of individual cells expressing phrA signaling within and across droplets. Currently, we are determining the effect of environmental conditions such as oxygen and carbohydrates on phrA-dependent signaling. 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.


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