(2mj) Extending a Microfluidic Platform to Elucidate Bacterial Communication in humans its impact on disease

Keywords: microfluidics, single cell analysis, cell-cell communication, human vaginal microbiome, Streptococcus pneumoniae, infectious disease

Summary:

Bacteria are ten times more abundant than host cells in the human body and their interactions

between each other and the host cells are often complex, leading to life-threatening infections that cause diseases. For instance, Streptococcus pneumoniae (Spn), a major human pathogen that causes over a million annual deaths in young children and the elderly, worldwide, also colonizes the human nasopharynx of many humans without causing any symptoms in its commensal form. In addition, the displacement of vaginal lactobacilli by strict anaerobic bacteria in the vagina is characteristic of bacterial vaginosis (BV), the leading cause of healthcare visits for women of reproductive age. Many questions remain about how bacteria switch from being a commensal to a pathogenic strain. To answer these questions, I adapted a microfluidic platform that enables high- parallel cultivation of bacteria in order to dissect and characterize microbial interactions in droplets using a water-in-oil emulsion that creates thousands of monodisperse mini-bioreactors.

During my dissertation, my goal was to develop a proof of concept to demonstrate microdroplets as an effective tool for co-culturing and recapitulating negative interactions between vaginal bacteria. My results recapitulated the killing effect of a health-promoting vaginal bacterium, against two vaginal putative pathogens, both in individual and pooled droplets. This project was the first to demonstrate co-cultivation and interactions of vaginal bacteria in droplets, used pneumatic sorting to isolate individual droplets to count cells, and presented a new technique that used less chemical reagent, lower costs, and high-parallel experiments to study microbial interactions in the vagina. The goal of my second project determined the effect of limited iron availability on growth of Lactobacillus crispatus and L. iners. I found that limited iron decreases growth of L. iners, but has no effect on growth of L. crispatus. These findings raise questions as to the mechanisms for iron sequestration for L. iners and L. crispatus, how they survive during menses when iron becomes available, and the influence of iron uptake on population dynamics. I also developed a method to pool vaginal fluid based on similar vaginal microflora and I discovered that L. iners grew in L. crispatus-dominated vaginal fluid. My work developed a novel method for culturing a hard to grow bacterium, ex vivo using sterile-filtered human vaginal fluid, and developed a model system simulating the human vagina, which has further implications for studying microbial interaction ex vivo.

During my postdoc, I studied 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. The overarching goal of my postdoc is to understand whether and how Spn cell-cell communication orchestrates this lifestyle switch. To answer this question, 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, a peptide regulated cell-cell communication system that promotes virulence in animal models of Spn carriage and pneumonia. 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. 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 deletion 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 captured substantial heterogeneity of individual cells expressing phrA within and across droplets. We are currently comparing signaling dynamics and heterogeneity for cells induced with native versus exogenously added peptides, as well as cells induced by signal from neighboring cells. In the long term, we hope to understand the kinetics of cell-signaling at the single cell level and exploit this knowledge for the development of anti-pneumococcal therapies.

Research interests

My research interests include using concepts in chemical engineering to answer biological questions. I am adapting a microfluidic platform to understand mechanisms that lead to diseases in the host. As a PI, I will design and develop screens that identify gene determinants for disease. I will develop model systems that simulate natural environments to culture and conduct competition experiments. I will also develop a microfluidic pipeline for investigating the effects that individual hosts have on mechanisms for disease.

Teaching interests

My teaching interests include all of the core classes in chemical engineering, especially material and energy balances, fluids, and transport phenomena. I am also interested in designing and teaching the course, biomedical applications for microfluidics.